Apolipoprotein A-I agonists and their use to treat dyslipidemic disorders

ABSTRACT

The present invention provides peptides and peptide analogues that mimic the structural and pharmacological properties of human ApoA-I. The peptides and peptide analogues are useful to treat a variety of disorders associated with dyslipidemia.

TABLE OF CONTENTS

1. INTRODUCTION

2. BACKGROUND OF THE INVENTION

2.1. CHOLESTEROL TRANSPORT

2.2. REVERSE CHOLESTEROL TRANSPORT

2.3. CURRENT TREATMENTS FOR DYSLIPOPROTEINEMIAS

2.4. ApoA-I AS A TARGET

3. SUMMARY OF THE INVENTION

3.1. ABBREVIATIONS

3.2. DEFINITIONS

4. BRIEF DESCRIPTION OF THE FIGURES

5. DETAILED DESCRIPTION OF THE INVENTION

5.1. PEPTIDE STRUCTURE AND FUNCTION

5.1.1. ANALYSIS OF STRUCTURE AND FUNCTION

5.1.2. PREFERRED EMBODIMENTS

5.2 SYNTHESIS AND PURIFICATION OF THE ApoA-I PEPTIDE AGONISTS

5.2.1 CHEMICAL SYNTHESIS

5.2.2 RECOMBINANT SYNTHESIS

5.2.3 PURIFICATION OF PEPTIDES

5.3 PHARMACEUTICAL FORMULATIONS AND METHODS OF TREATMENT

5.3.1 ApoA-I AGONISTS AND PEPTIDE/LIPID COMPLEX AS THE ACTIVE INGREDIENT

5.3.2 METHODS OF THE TREATMENT

5.3.3 PHARMACEUTICAL FORMULATIONS

5.4 OTHER USES

6. EXAMPLE: SYNTHESIS OF PEPTIDE AGONISTS OF ApoA-I

6.1 SYNTHESIS OF CORE PEPTIDES

6.2 SYNTHESIS OF PEPTIDE AMIDES

6.3 SYNTHESIS OF N-TERMINAL ACYLATED PEPTIDES

6.4 CLEAVAGE AND DEPROTECTION

6.5 PURIFICATION

6.6 CHARACTERIZATION

6.6.1 LC-MS

6.6.2 AMINO ACID ANALYSIS

6.7 SYNTHESIS OF BRANCHED NETWORKS

7 EXAMPLE: STRUCTURAL AND LIPID BINDING ANALYSIS OF ApoA-I PEPTIDES

7.1 CIRCULAR DICHROISM

7.1.1 EXPERIMENTAL METHOD

7.1.2 HELICITY DETERMINATION

7.1.3 RESULTS

7.2 FLUORESCENCE SPECTROSCOPY

7.2.1 FLUORESCENCE SPECTRAL ANALYSIS

7.2.2 RESULTS AND DISCUSSION

7.3 NUCLEAR MAGNETIC RESONANCE (NMR)

7.3.1 NMR SAMPLE PREPARATION

7.3.2 NMR SPECTROSCOPY

7.3.3 NMR ASSIGNMENT

7.3.4 RESULTS AND DISCUSSION

7.3.5 PEPTIDES CONTAINING INTERNAL GLYCINES ARE HELICAL IN THE PRESENCEOF TFE

8. EXAMPLE: LCAT ACTIVATION ASSAY

8.1 PREPARATION OF SUBSTRATE VESICLES

8.2 PURIFICATION OF LCAT

8.2.1 PREPARATION OF LPDS

8.2.2 PHENYLSEPHAROSE CHROMATOGRAPHY

8.2.3 AFFIGELBLUE CHROMATOGRAPHY

8.2.4 ConA CHROMATOGRAPHY

8.2.5 ANTI-ApoA-I AFFINITY CHROMATOGRAPHY

8.3 RESULTS

EXAMPLE: PHARMACOKINETICS OF THE ApoA-I AGONISTS

9.1. SYNTHESIS OF RADIOLABELED PEPTIDES

9.2. PHARMACOKINETICS IN MICE

9.3. STABILITY IN HUMAN SERUM

9.3.1. EXPERIMENTAL METHODS

9.3.2. RESULTS

9.4. FORMATION OF PRE-βLIKE PARTICLES

9.4.1. EXPERIMENTAL METHOD

9.4.2. RESULTS

9.5. ASSOCIATION OF PEPTIDE 4 WITH HUMAN LIPOPROTEINS

9.5.1. EXPERIMENTAL METHODS

9.5.2. RESULTS

9.6. PEPTIDE 4 SELECTIVELY BINDS HDL LIPIDS IN HUMAN PLASMA

9.6.1. EXPERIMENTAL METHOD

9.6.2. RESULTS

10. EXAMPLE: THE ApoA-I AGONISTS PROMOTE CHOLESTEROL EFFLUX

11. EXAMPLE: USE OF THE ApoA-I AGONISTS IN ANIMAL MODEL SYSTEMS

11.1. PREPARATION OF THE PHOSPHOLIPID/PEPTIDE COMPLEXES

11.2. ISOLATION AND CHARACTERIZATION OF THE PEPTIDE/PHOSPHOLIPIDPARTICLES

11.3. INJECTION IN THE RABBIT

11.4. ANALYSIS OF THE RABBIT SERA

11.5. RESULTS

12. EXAMPLE: PREPARATION OF PEPTIDE-LIPID COMPLEX BY CO-LYOPHILIZATIONAPPROACH

12.1. CHARACTERIZATION OF COMPLEXES BY SUPEROSE 6 GEL FILTRATIONCHROMATOGRAPHY

12.2. SUPEROSE 6 GEL FILTRATION CHROMATOGRAPHY OF HUMAN HDL

13. EXAMPLE: PREPARATION OF ANTIBODIES

1. INTRODUCTION

The invention relates to apolipoprotein A-I (ApoA-I) agonistcompositions for treating disorders associated with dyslipoproteinemia,including hypercholesterolemia, cardiovascular disease, atherosclerosis,restenosis, and other disorders such as septic shock.

2. BACKGROUND OF THE INVENTION

Circulating cholesterol is carried by plasma lipoproteins--particles ofcomplex lipid and protein composition that transport lipids in theblood. Low density lipoproteins (LDL), and high density lipoproteins(HDL) are the major cholesterol carriers. LDL are believed to beresponsible for the delivery of cholesterol from the liver (where it issynthesized or obtained from dietary sources) to extrahepatic tissues inthe body. The term "reverse cholesterol transport" describes thetransport of cholesterol from extrahepatic tissues to the liver where itis catabolized and eliminated. It is believed that plasma HDL particlesplay a major role in the reverse transport process, acting as scavengersof tissue cholesterol.

The evidence linking elevated serum cholesterol to coronary heartdisease is overwhelming. For example, atherosclerosis is a slowlyprogressive disease characterized by the accumulation of cholesterolwithin the arterial wall. Compelling evidence supports the concept thatlipids deposited in atherosclerotic lesions are derived primarily fromplasma LDL; thus, LDLs have popularly become known as the "bad"cholesterol. In contrast, HDL serum levels correlate inversely withcoronary heart disease--indeed, high serum levels of HDL are regarded asa negative risk factor. It is hypothesized that high levels of plasmaHDL are not only protective against coronary artery disease, but mayactually induce regression of atherosclerotic plaques (e.g., see Badimonet al., 1992, Circulation 86 (Suppl. III):86-94). Thus, HDL havepopularly become known as the "good" cholesterol.

2.1. CHOLESTEROL TRANSPORT

The fat-transport system can be divided into two pathways: an exogenousone for cholesterol and triglycerides absorbed from the intestine, andan endogenous one for cholesterol and triglycerides entering thebloodstream from the liver and other non-hepatic tissue.

In the exogenous pathway, dietary fats are packaged into lipoproteinparticles called chylomicrons which enter the bloodstream and delivertheir triglycerides to adipose tissue (for storage) and to muscle (foroxidation to supply energy). The remnant of the chylomicron, containingcholesteryl esters, is removed from the circulation by a specificreceptor found only on liver cells. This cholesterol then becomesavailable again for cellular metabolism or for recycling to extrahepatictissues as plasma lipoproteins.

In the endogenous pathway, the liver secretes a large, very-low-densitylipoprotein particle (VLDL) into the bloodstream. The core of VLDLsconsists mostly of triglycerides synthesized in the liver, with asmaller amount of cholesteryl esters (either synthesized in the liver orrecycled from chylomicrons). Two predominant proteins are displayed onthe surface of VLDLs, apoprotein B-100 and apoprotein E. When a VLDLreaches the capillaries of adipose tissue or of muscle, itstriglycerides are extracted resulting in a new kind of particle,decreased in size and enriched in cholesteryl esters but retaining itstwo apoproteins, called intermediate-density lipoprotein (IDL).

In human beings, about half of the IDL particles are removed from thecirculation quickly (within two to six hours of their formation),because they bind tightly to liver cells which extract their cholesterolto make new VLDL and bile acids. The IDL particles which are not takenup by the liver remain in the circulation longer. In time, theapoprotein E dissociates from the circulating particles, converting themto LDL having apoprotein B-100 as their sole protein.

Primarily, the liver takes up and degrades most of the cholesterol tobile acids, which are the end products of cholesterol metabolism. Theuptake of cholesterol containing particles is mediated by LDL receptors,which are present in high concentrations on hepatocytes. The LDLreceptor binds both apoprotein E and apoprotein B-100, and isresponsible for binding and removing both IDLs and LDLs from thecirculation. However, the affinity of apoprotein E for the LDL receptoris greater than that of apoprotein B-100. As a result, the LDL particleshave a much longer circulating life span than IDL particles--LDLscirculate for an average of two and a half days before binding to theLDL receptors in the liver and other tissues. High serum levels of LDL(the "bad" cholesterol) are positively associated with coronary heartdisease. For example, in atherosclerosis, cholesterol derived fromcirculating LDLs accumulates in the walls of arteries leading to theformation of bulky plaques that inhibit the flow of blood until a cloteventually forms, obstructing the artery causing a heart attack orstroke.

Ultimately, the amount of intracellular cholesterol liberated from theLDLs controls cellular cholesterol metabolism. The accumulation ofcellular cholesterol derived from VLDLs and LDLs controls threeprocesses: first, it reduces cellular cholesterol synthesis by turningoff the synthesis of HMGCoA reductase--a key enzyme in the cholesterolbiosynthetic pathway. Second, the incoming LDL-derived cholesterolpromotes storage of cholesterol by activating ACAT--the cellular enzymewhich converts cholesterol into cholesteryl esters that are deposited instorage droplets. Third, the accumulation of cholesterol within the celldrives a feedback mechanism that inhibits cellular synthesis of new LDLreceptors. Cells, therefore, adjust their complement of LDL receptors sothat enough cholesterol is brought in to meet their metabolic needs,without overloading. (For a review, see Brown & Goldstein, In, ThePharmacological Basis Of Therapeutics, 8th Ed., Goodman & Gilman,Pergamon Press, N.Y., 1990, Ch. 36, pp. 874-896).

2.2. REVERSE CHOLESTEROL TRANSPORT

In sum, peripheral (non-hepatic) cells obtain their cholesterol from acombination of local synthesis and the uptake of preformed sterol fromVLDLs and LDLs. In contrast, reverse cholesterol transport (RCT) is thepathway by which peripheral cell cholesterol can be returned to theliver for recycling to extrahepatic tissues, or excretion into theintestine in bile, either in modified or in oxidized form as bile acids.The RCT pathway represents the only means of eliminating cholesterolfrom most extrahepatic tissues, and is crucial to maintenance of thestructure and function of most cells in the body.

The RCT consists mainly of three steps: (a) cholesterol efflux, theinitial removal of cholesterol from various pools of peripheral cells;(b) cholesterol esterification by the action of lecithin:cholesterolacyltransferase (LCAT), preventing a re-entry of effluxed cholesterolinto cells; and (c) uptake/delivery of HDL cholesteryl ester to livercells. The RCT pathway is mediated by HDLs. HDL is a generic term forlipoprotein particles which are characterized by their high density. Themain lipidic constituents of HDL complexes are various phospholipids,cholesterol (ester) and triglycerides. The most prominent apolipoproteincomponents are A-I and A-II which determine the functionalcharacteristics of HDL; furthermore minor amounts of apolipoprotein C-I,C-II, C-III, D, E, J, etc. have been observed. HDL can exist in a widevariety of different sizes and different mixtures of the above-mentionedconstituents depending on the status of remodeling during the metabolicRCT cascade.

The key enzyme involved in the RCT pathway is LCAT. LCAT is producedmainly in the liver and circulates in plasma associated with the HDLfraction. LCAT converts cell derived cholesterol to cholesteryl esterswhich are sequestered in HDL destined for removal. Cholesteryl estertransfer protein (CETP) and phospholipid transfer protein (PLTP)contribute to further remodeling the circulating HDL population. CETPcan move cholesteryl esters made by LCAT to other lipoproteins,particularly ApoB-containing lipoproteins, such as VLDL and LDL. PLTPsupplies lecithin to HDL. HDL triglycerides can be catabolized by theextracellular hepatic triglyceride lipase, and lipoprotein cholesterolis removed by the liver via several mechanisms.

Each HDL particle contains at least one copy (and usually two to fourcopies) of ApoA-I. ApoA-I is synthesized by the liver and smallintestine as preproapolipoprotein which is secreted as a proprotein thatis rapidly cleaved to generate a mature polypeptide having 243 aminoacid residues. ApoA-I consists mainly of 6 to 8 different 22 amino acidrepeats spaced by a linker moiety which is often proline, and in somecases consists of a stretch made up of several residues. ApoA-I formsthree types of stable complexes with lipids: small, lipid-poor complexesreferred to as pre-beta-1 HDL; flattened discoidal particles containingpolar lipids (phospholipid and cholesterol) referred to as pre-beta-2HDL; and spherical particles containing both polar and nonpolar lipids,referred to as spherical or mature HDL (HDL₃ and HDL₂). Most HDL in thecirculating population contain both ApoA-I and ApoA-II (the second majorHDL protein) and are referred to herein as the AI/AII-HDL fraction ofHDL. However, the fraction of HDL containing only ApoA-I (referred toherein as the AI-HDL fraction) appear to be more effective in RCT.Certain epidemiologic studies support the hypothesis that the AI-HDLfraction is anti-atherogenic. (Parra et al., 1992, Arterioscler. Thromb.12:701-707; Decossin et al., 1997, Eur. J. Clin. Invest. 27:299-307).

Although the mechanism for cholesterol transfer from the cell surface(i.e., cholesterol efflux) is unknown, it is believed that thelipid-poor complex, pre-beta-1 HDL is the preferred acceptor forcholesterol transferred from peripheral tissue involved in RCT. (SeeDavidson et al., 1994, J. Biol. Chem. 269:22975-22982; Bielicki et al.,1992, J. Lipid Res. 33:1699-1709; Rothblat et al., 1992, J. Lipid Res.33:1091-1097; and Kawano et al., 1993, Biochemistry 32:5025-5028; Kawanoet al., 1997, Biochemistry 36:9816-9825). During this process ofcholesterol recruitment from the cell surface, pre-beta-1 HDL is rapidlyconverted to pre-beta-2 HDL. PLTP may increase the rate of pre-beta-2disc formation, but data indicating a role for PLTP in RCT is lacking.LCAT reacts preferentially with discoidal and spherical HDL,transferring the 2-acyl group of lecithin or other phospholipids to thefree hydroxyl residue of cholesterol to generate cholesteryl esters(retained in the HDL) and lysolecithin. The LCAT reaction requiresApoA-I as activator; i.e., ApoA-I is the natural cofactor for LCAT. Theconversion of cholesterol to its ester sequestered in the HDL preventsre-entry of cholesterol into the cell, the result being that cholesterylesters are destined for removal. Cholesteryl esters in the mature HDLparticles in the AI-HDL fraction (i.e., containing ApoA-I and noApoA-II) are removed by the liver and processed into bile moreeffectively than those derived from HDL containing both ApoA-I andApoA-II (the AI/AII-HDL fraction). This may be due, in part, to the moreeffective binding of AI-HDL to the hepatocyte membrane. The existence ofan HDL receptor has been hypothesized, and recently a scavengerreceptor, SR-BI, was identified as an HDL receptor (Acton et al., 1996,Science 271:518-520; Xu et al., 1997, Lipid Res. 38:1289-1298). TheSR-BI is expressed most abundantly in steroidogenic tissues (e.g., theadrenals), and in the liver (Landshulz et al., 1996, J. Clin. Invest.98:984-995; Rigotti et al., 1996, J. Biol. Chem. 271:33545-33549).

CETP does not appear to play a major role in RCT, and instead isinvolved in the metabolism of VLDL-and LDL-derived lipids. However,changes in CETP activity or its acceptors, VLDL and LDL, play a role in"remodeling" the HDL population. For example, in the absence of CETP,the HDLs become enlarged particles which are not cleared. (For reviewson RCT and HDLs, see Fielding & Fielding, 1995, J. Lipid Res.36:211-228; Barrans et al., 1996, Biochem. Biophys. Acta. 1300:73-85;Hirano et al., 1997, Arterioscler. Thromb. Vasc. Biol. 17(6):1053-1059).

2.3. CURRENT TREATMENTS FOR DYSLIPOPROTEINEMIAS

A number of treatments are currently available for lowering serumcholesterol and triglycerides (see, e.g., Brown & Goldstein, supra).However, each has its own drawbacks and limitations in terms ofefficacy, side-effects and qualifying patient population.

Bile-acid-binding resins are a class of drugs that interrupt therecycling of bile acids from the intestine to the liver; e.g.,cholestyramine (Questran Light®, Bristol-Myers Squibb), and colestipolhydrochloride (Colestid®, The Upjohn Company). When taken orally, thesepositively-charged resins bind to the negatively charged bile acids inthe intestine. Because the resins cannot be absorbed from the intestine,they are excreted carrying the bile acids with them. The use of suchresins, however, at best only lowers serum cholesterol levels by about20%, and is associated with gastrointestinal side-effects, includingconstipation and certain vitamin deficiencies. Moreover, since theresins bind other drugs, other oral medications must be taken at leastone hour before or four to six hours subsequent to ingestion of theresin; thus, complicating heart patient's drug regimens.

The statins are cholesterol lowering agents that block cholesterolsynthesis by inhibiting HMGCoA reductase--the key enzyme involved in thecholesterol biosynthetic pathway. The statins, e.g., lovastatin(Mevacor®, Merck & Co., Inc.), and pravastatin (Pravachol®,Bristol-Myers Squibb Co.) are sometimes used in combination withbile-acid-binding resins. The statins significantly reduce serumcholesterol and LDL-serum levels, and slow progression of coronaryatherosclerosis. However, serum HDL cholesterol levels are onlymoderately increased. The mechanism of the LDL lowering effect mayinvolve both reduction of VLDL concentration and induction of cellularexpression of LDL-receptor, leading to reduced production and/orincreased catabolism of LDLs. Side effects, including liver and kidneydysfunction are associated with the use of these drugs (Physicians DeskReference, Medical Economics Co., Inc., Montvale, N.J., 1997). Recently,the FDA has approved atorvastatin (an HMGCoA reductase inhibitordeveloped by Parke-Davis) (Warner Lambert) for the market to treat rarebut urgent cases of familial hypercholesterolemia (1995, Scrip20(19):10).

Niacin, or nicotinic acid, is a water soluble vitamin B-complex used asa dietary supplement and antihyperlipidemic agent. Niacin diminishesproduction of VLDL and is effective at lowering LDL. In some cases, itis used in combination with bile-acid binding resins. Niacin canincrease HDL when used at adequate doses, however, its usefulness islimited by serious side effects when used at such high doses.

Fibrates are a class of lipid-lowering drugs used to treat various formsof hyperlipidemia (i.e., elevated serum triglycerides) which may also beassociated with hypercholesterolemia. Fibrates appear to reduce the VLDLfraction and modestly increase HDL--however the effects of these drugson serum cholesterol is variable. In the United States, fibrates havebeen approved for use as antilipidemic drugs, but have not receivedapproval as hypercholesterolemia agents. For example, clofibrate(Atromid-S®, Wyeth-Ayerst Laboratories) is an antilipidemic agent whichacts (via an unknown mechanism) to lower serum triglycerides by reducingthe VLDL fraction. Although serum cholesterol may be reduced in certainpatient subpopulations, the biochemical response to the drug isvariable, and is not always possible to predict which patients willobtain favorable results. Atromid-S® has not been shown to be effectivefor prevention of coronary heart disease. The chemically andpharmacologically related drug, gemfibrozil (Lopid®, Parke-Davis) is alipid regulating agent which moderately decreases serum triglyceridesand VLDL cholesterol, and moderately increases HDL cholesterol--the HDL₂and HDL₃ subfractions as well as both ApoA-I and A-II (i.e., theAI/AII-HDL fraction). However, the lipid response is heterogeneous,especially among different patient populations. Moreover, whileprevention of coronary heart disease was observed in male patientsbetween 40-55 without history or symptoms of existing coronary heartdisease, it is not clear to what extent these findings can beextrapolated to other patient populations (e.g., women, older andyounger males). Indeed, no efficacy was observed in patients withestablished coronary heart disease. Serious side-effects are associatedwith the use of fibrates including toxicity such as malignancy,(especially gastrointestinal cancer), gallbladder disease and anincreased incidence in non-coronary mortality. These drugs are notindicated for the treatment of patients with high LDL or low HDL astheir only lipid abnormality (Physician's Desk Reference, 1997, MedicalEconomics Co., Inc. Montvale, N.J.).

Oral estrogen replacement therapy may be considered for moderatehypercholesterolemia in post-menopausal women. However, increases in HDLmay be accompanied with an increase in triglycerides. Estrogen treatmentis, of course, limited to a specific patient population (postmenopausalwomen) and is associated with serious side effects including inductionof malignant neoplasms, gall bladder disease, thromboembolic disease,hepatic adenoma, elevated blood pressure, glucose intolerance, andhypercalcemia.

Thus, there is a need to develop safer drugs that are efficacious inlowering serum cholesterol, increasing HDL serum levels, preventingcoronary heart disease, and/or treating existing disease, especiallyatherosclerosis.

2.4. ApoA-I AS A TARGET

None of the currently available drugs for lowering cholesterol safelyelevate HDL levels and stimulate RCT--most appear to operate on thecholesterol transport pathway, modulating dietary intake, recycling,synthesis of cholesterol, and the VLDL population.

While it is desirable to find drugs that stimulate cholesterol effluxand removal, several potential targets in the RCT exist--e.g., LCAT, HDLand its various components (ApoA-I, ApoA-II and phospholipids), PLTP,and CETP--and it is not known which target would be most effective atachieving desirable lipoprotein profiles and protective effects.Perturbation of any single component in the RCT pathway ultimatelyaffects the composition of circulating lipoprotein populations, and theefficiency of RCT.

Several lines of evidence based on data obtained in vivo implicate theHDL and its major protein component, ApoA-I, in the prevention ofatherosclerotic lesions, and potentially, the regression ofplaques--making these attractive targets for therapeutic intervention.First, an inverse correlation exists between serum ApoA-I (HDL)concentration and atherogenesis in man (Gordon & Rifkind, 1989, N. Eng.J. Med. 321:1311-1316; Gordon et al., 1989, Circulation 79:8-15).Indeed, specific subpopulations of HDL have been associated with areduced risk for atherosclerosis in humans (Miller, 1987, Amer. Heart113:589-597; Cheung et al., 1991, Lipid Res. 32:383-394); Fruchart &Ailhaud, 1992, Clin. Chem. 38:79).

Second, animal studies support the protective role of ApoA-I (HDL).Treatment of cholesterol fed rabbits with ApoA-I or HDL reduced thedevelopment and progression of plaque (fatty streaks) in cholesterol-fedrabbits. (Koizumi et al., 1988, J. Lipid Res. 29:1405-1415; Badimon etal., 1989, Lab. Invest. 60:455-461; Badimon et al., 1990, J. Clin.Invest. 85:1234-1241). However, the efficacy varied depending upon thesource of HDL (Beitz et al., 1992, Prostaglandins, Leukotrienes andEssential Fatty Acids 47:149-152; Mezdour et al., 1995, Atherosclerosis113:237-246).

Third, direct evidence for the role of ApoA-I was obtained fromexperiments involving transgenic animals. The expression of the humangene for ApoA-I transferred to mice genetically predisposed todiet-induced atherosclerosis protected against the development of aorticlesions (Rubin et al., 1991, Nature 353:265-267). The ApoA-I transgenewas also shown to suppress atherosclerosis in ApoE-deficient mice and inApo(a) transgenic mice (Paszty et al., 1994, J. Clin. Invest.94:899-903; Plump et al., 1994, Proc. Natl. Acad. Sci. USA 91:9607-9611;Liu et al., 1994, J. Lipid Res. 35:2263-2266). Similar results wereobserved in transgenic rabbits expressing human ApoA-I (Duverger, 1996,Circulation 94:713-717; Duverger et al., 1996, Arterioscler. Thromb.Vasc. Biol. 16:1424-1429), and in transgenic rats where elevated levelsof human ApoA-I protected against atherosclerosis and inhibitedrestenosis following balloon angioplasty (Burkey et al., 1992,Circulation, Supplement I, 86:I-472, Abstract No. 1876; Burkey et al.,1995, J. Lipid Res. 36:1463-1473).

The AI-HDL appear to be more efficient at RCT than the AI/AII-HDLfraction. Studies with mice transgenic for human ApoA-I or Apo-I andApoA-II (AI/AII) showed that the protein composition of HDLsignificantly affects its role--AI-HDL is more anti-atherogenic thanAI/AII-HDL (Schultz et al., 1993, Nature 365:762-764). Parallel studiesinvolving transgenic mice expressing the human LCAT gene demonstratethat moderate increases in LCAT activity significantly changelipoprotein cholesterol levels, and that LCAT has a significantpreference for HDL containing ApoA-I (Francone et al., 1995, J. Clinic.Invest. 96:1440-1448; Berard et al., 1997, Nature Medicine3(7):744-749). While these data support a significant role for ApoA-I inactivating LCAT and stimulating RCT, additional studies demonstrate amore complicated scenario: a major component of HDL that modulatesefflux of cell cholesterol is the phospholipids (Fournier et al., 1996,J. Lipid Res. 37:1704-1711).

In view of the potential role of HDL, i.e., both ApoA-I and itsassociated phospholipid, in the protection against atheroscleroticdisease, human clinical trials utilizing recombinantly produced ApoA-Iwere commenced, discontinued and apparently re-commenced by UCB Belgium(Pharmaprojects, Oct. 27, 1995; IMS R&D Focus, Jun. 30, 1997; DrugStatus Update, 1997, Atherosclerosis 2(6):261-265); see also M. Erikssonat Congress, "The Role of HDL in Disease Prevention," Nov. 7-9, 1996,Fort Worth; Lacko & Miller, 1997, J. Lip. Res. 38:1267-1273; andWO94/13819) and were commenced and discontinued by Bio-Tech(Pharmaprojects, Apr. 7, 1989). Trials were also attempted using ApoA-Ito treat septic shock (Opal, "Reconstituted HDL as a Treatment Strategyfor Sepsis," IBC's 7th International Conference on Sepsis, Apr. 28-30,1997, Washington, D.C.; Gouni et al., 1993, J. Lipid Res. 94:139-146;Levine, WO96/04916). However, there are many pitfalls associated withthe production and use of ApoA-I, making it less than ideal as a drug;e.g., ApoA-I is a large protein that is difficult and expensive toproduce; significant manufacturing and reproducibility problems must beovercome with respect to stability during storage, delivery of an activeproduct and half-life in vivo.

In view of these drawbacks, attempts have been made to prepare peptidesthat mimic ApoA-I. Since the key activities of ApoA-I have beenattributed to the presence of multiple repeats of a unique secondarystructural feature in the protein--a class A amphipathic α-helix(Segrest, 1974, FEBS Lett. 38:247-253), most efforts to design peptideswhich mimic the activity of ApoA-I have focused on designing peptideswhich form class A-type amphipathic α-helices.

Class A-type amphipathic α-helices are unique in that positively chargedamino acid residues are clustered at the hydrophobic-hydrophilicinterface and negatively charged amino acid residues are clustered atthe center of the hydrophilic face. Furthermore, class A α-helicalpeptides have a hydrophobic angle of less than 180° (Segrest et al.,1990, PROTEINS: Structure, Function and Genetics 8:103-117). The initialde novo strategies to design ApoA-I mimics were not based upon theprimary sequences of naturally occurring apolipoproteins, but ratherupon incorporating these unique Class A helix features into thesequences of the peptide analogues, as well as some of the properties ofthe ApoA-I domains (see, e.g., Davidson et al., 1996, Proc. Natl. Acad.Sci. USA 93:13605-13610; Rogers et al., 1997, Biochemistry 36:288-300;Lins et al., 1993, Biochim. Biophys. Acta biomembranes 1151:137-142; Jiand Jonas, 1995, J. Biol. Chem. 270:11290-11297; Collet et al., 1997,Journal of Lipid Research, 38:634-644; Sparrow and Gotto, 1980, Ann.N.Y. Acad. Sci. 348:187-211; Sparrow and Gotto, 1982, CRC Crit. Rev.Biochem. 13:87-107; Sorci-Thomas et al., 1993, J. Biol. Chem.268:21403-21409; Wang et al., 1996, Biochim. Biophys. Acta 174-184;Minnich et al., 1992, J. Biol. Chem. 267:16553-16560; Holvoet et al.,1995, Biochemistry 34:13334-13342; Sorci-Thomas et al., 1997, J. Biol.Chem. 272(11):7278-7284; and Frank et al., 1997, Biochemistry36:1798-1806).

In one study, Fukushima et al. synthesized a 22-residue peptide composedentirely of Glu, Lys and Leu residues arranged periodically so as toform an amphipathic α-helix with equal hydrophilic and hydrophobic faces("ELK peptide") (Fukushima et al., 1979, J. Amer. Chem. Soc.101(13):3703-3704; Fukushima et al., 1980, J. Biol. Chem.255:10651-10657). The ELK peptide shares 41% sequence homology with the198-219 fragment of ApoA-I. As studied by quantitative ultrafiltration,gel permeation chromatography and circular dichroism, this ELK peptidewas shown to effectively associate with phospholipids and mimic some ofthe physical and chemical properties of ApoA-I (Kaiser et al., 1983,Proc. Natl. Acad. Sci. USA 80:1137-1140; Kaiser et al., 1984, Science223:249-255; Fukushima et al., 1980, supra; Nakagawa et al., 1985, J.Am. Chem. Soc. 107:7087-7092). Yokoyama et al. concluded from suchstudies that the crucial factor for LCAT activation is simply thepresence of a large enough amphipathic structure (Yokoyama et al., 1980,J. Biol. Chem. 255(15):7333-7339). A dimer of this 22-residue peptidewas later found to more closely mimic ApoA-I than the monomer; based onthese results, it was suggested that the 44-mer, which is punctuated inthe middle by a helix breaker (either Gly or Pro), represented theminimal functional domain in ApoA-I (Nakagawa et al., 1985, supra).

Another study involved model amphipathic peptides called "LAP peptides"(Pownall et al., 1980, Proc. Natl. Acad. Sci. USA 77(6):3154-3158;Sparrow et al., 1981, In: Peptides: Synthesis-Structure-Function, Rochand Gross, Eds., Pierce Chem. Co., Rockford, Ill., 253-256). Based onlipid binding studies with fragments of native apolipoproteins, severalLAP peptides were designed, named LAP-16, LAP-20 and LAP-24 (containing16, 20 and 24 amino acid residues, respectively). These modelamphipathic peptides share no sequence homology with the apolipoproteinsand were designed to have hydrophilic faces organized in a manner unlikethe class A-type amphipathic helical domains associated withapolipoproteins (Segrest et al., 1992, J. Lipid Res. 33:141-166). Fromthese studies, the authors concluded that a minimal length of 20residues is necessary to confer lipid-binding properties to modelamphipathic peptides.

Studies with mutants of LAP20 containing a proline residue at differentpositions in the sequence indicated that a direct relationship existsbetween lipid binding and LCAT activation, but that the helicalpotential of a peptide alone does not lead to LCAT activation (Ponsin etal., 1986 J. Biol. Chem. 261(20):9202-9205). Moreover, the presence ofthis helix breaker (Pro) close to the middle of the peptide reduced itsaffinity for phospholipid surfaces as well as its ability to activateLCAT. While certain of the LAP peptides were shown to bind phospholipids(Sparrow et al., supra), controversy exists as to the extent to whichLAP peptides are helical in the presence of lipids (Buchko et al., 1996,J. Biol. Chem. 271(6):3039-3045; Zhong et al., 1994, Peptide Research7(2):99-106).

Segrest et al. have synthesized peptides composed of 18 to 24 amino acidresidues that share no sequence homology with the helices of ApoA-I(Kannelis et al., 1980, J. Biol. Chem. 255(3):11464-11472; Segrest etal., 1983, J. Biol. Chem. 258:2290-2295). The sequences werespecifically designed to mimic the amphipathic helical domains of classA exchangeable apolipoproteins in terms of hydrophobic moment (Eisenberget al., 1982, Nature 299:371-374) and charge distribution (Segrest etal., 1990, Proteins 8:103-117; U.S. Pat. No. 4,643,988). One 18-residuepeptide, the "18A" peptide, was designed to be a model class-A α-helix(Segrest et al., 1990, supra). Studies with these peptides and otherpeptides having a reversed charged distribution, like the "18R" peptide,have consistently shown that charge distribution is critical foractivity; peptides with a reversed charge distribution exhibit decreasedlipid affinity relative to the 18A class-A mimics and a lower helicalcontent in the presence of lipids (Kanellis et al., 1980, J. Biol. Chem.255:11464-11472; Anantharamaiah et al., 1985, J. Biol. Chem.260:10248-10255; Chung et al., 1985, J. Biol. Chem. 260:10256-10262;Epand et al., 1987, J. Biol. Chem. 262:9389-9396; Anantharamaiah et al.,1991, Adv. Exp. Med. Biol. 285:131-140).

Other synthetic peptides sharing no sequence homology with theapolipoproteins which have been proposed with limited success includedimers and trimers of the 18A peptide (Anantharamaiah et al., 1986,Proteins of Biological Fluids 34:63-66), GALA and EALA peptides(Subbarao et al., 1988, PROTEINS: Structure, Function and Genetics3:187-198) and ID peptides (Labeur et al., 1997, Arteriosclerosis,Thrombosis and Vascular Biology 17:580-588) and the 18AM4 peptide(Brasseur et al., 1993, Biochim. Biophys. Acta 1170:1-7).

A "consensus" peptide containing 22-amino acid residues based on thesequences of the helices of human ApoA-I has also been designed(Anantharamaiah et al., 1990, Arteriosclerosis 10(1):95-105;Venkatachalapathi et al., 1991, Mol. Conformation and Biol.Interactions, Indian Acad. Sci. B:585-596). The sequence was constructedby identifying the most prevalent residue at each position of thehypothesized helices of human ApoA-I. Like the peptides described above,the helix formed by this peptide has positively charged amino acidresidues clustered at the hydrophilic-hydrophobic interface, negativelycharged amino acid residues clustered at the center of the hydrophilicface and a hydrophobic angle of less than 180°. While a dimer of thispeptide is somewhat effective in activating LCAT, the monomer exhibitedpoor lipid binding properties (Venkatachalapathi et al., 1991, supra).

Based primarily on in vitro studies with the peptides described above, aset of "rules" has emerged for designing peptides which mimic thefunction of apoA-I. Significantly, it is thought that an amphipathicα-helix having positively charged residues clustered at thehydrophilic-hydrophobic interface and negatively charged amino acidresidues clustered at the center of the hydrophilic face is required forlipid affinity and LCAT activation (Venkatachalapathi et al., 1991,supra). Anantharamaiah et al. have also indicated that the negativelycharged Glu residue at position 13 of the consensus 22-mer peptide,which is positioned within the hydrophobic face of the α-helix, plays animportant role in LCAT activation (Anantharamaiah et al., 1991, supra).Furthermore, Brasseur has indicated that a hydrophobic angle (pho angle)of less than 180° is required for optimal lipid-apolipoprotein complexstability, and also accounts for the formation of discoidal particleshaving the peptides around the edge of the lipid bilayer (Brasseur,1991, J. Biol. Chem. 66(24):16120-16127). Rosseneu et al. have alsoinsisted that a hydrophobic angle of less than 180° is required for LCATactivation (WO93/25581).

However, despite these "rules" to date, no one has designed or produceda peptide as active as ApoA-I--the best having less than 40% of theactivity of ApoA-I as measured by the LCAT activation assay describedherein. None of the peptide "mimetics" described in the literature havebeen demonstrated to be useful as a drug.

In view of the foregoing, there is a need for the development of astable ApoA-I agonist that mimics the activity of ApoA-I and which isrelatively simple and cost-effective to produce. However, the "rules"for designing efficacious ApoA-I mimetics have not been unraveled andthe principles for designing organic molecules with the function ofApoA-I are unknown.

3. SUMMARY OF THE INVENTION

The invention relates to ApoA-I agonists capable of forming amphipathicα-helices that mimic the activity of ApoA-I, with specific activities,i.e., units of activity (activation of LCAT)/unit of mass), approachingor exceeding that of the native molecule. In particular, the ApoA-Iagonists of the invention are peptides or peptide analogues that: formamphipathic helices (in the presence of lipids), bind lipids, formpre-β-like or HDL-like complexes, activate LCAT, increase serum levelsof HDL fractions, and promote cholesterol efflux.

The invention is based, in part, on the applicants' design and discoveryof peptides that mimic the function of ApoA-I. The peptides of theinvention were designed based on the supposed helical structure andamphipathic properties of the 22 amino acid consensus sequence which wasderived from the helical repeats of ApoA-I. Surprisingly, the peptidesof the invention have a specific activity well above that reported forApoA-I-derived peptides described in the literature. Indeed, someembodiments of the invention approach 100% of the activity of nativeApoA-I, whereas superagonists described herein exceed the specificactivity of ApoA-I.

The invention is illustrated by way of working examples that describethe structure, preparation and use of particular amphipathic peptidesthat form helices (in the presence of lipids), bind lipids, formcomplexes and increase LCAT activity. Based upon the structure andactivity of the exemplified embodiments, the applicants have devised aset of "rules" which can be used to design altered or mutated forms thatare also within the scope of the invention.

The invention also relates to pharmaceutical formulations containingsuch ApoA-I agonists (either as peptides or peptide-lipid complexes) asthe active ingredient, as well as methods for preparing suchformulations and their use to treat diseases associated withdyslipoproteinemia (e.g., cardiovascular diseases, atherosclerosis,metabolic syndrome), restenosis, or endotoxemia (e.g., septic shock).

3.1. ABBREVIATIONS

As used herein, the abbreviations for the genetically encodedL-enantiomeric amino acids are conventional and are as follows:

    ______________________________________                                                     One-Letter     Common                                                                         Amino Acid Symbol Abbreviation                   ______________________________________                                        Alanine      A              Ala                                                 Arginine R Arg                                                                Asparagine N Asn                                                              Aspartic acid D Asp                                                           Cysteine C Cys                                                                Glutamine Q Gln                                                               Glutamic acid E Glu                                                           Glycine G Gly                                                                 Histidine H His                                                               Isoleucine I Ile                                                              Leucine L Leu                                                                 Lysine K Lys                                                                  Methionine M Met                                                              Phenylalanine F Phe                                                           Proline P Pro                                                                 Serine S Ser                                                                  Threonine T Thr                                                               Tryptophan W Trp                                                              Tyrosine Y Tyr                                                                Valine V Val                                                                ______________________________________                                    

The abbreviations used for the D-enantiomers of the genetically encodedamino acids are lower-case equivalents of the one-letter symbols. Forexample, "R" designates L-arginine and "r" designates D-arginine.

3.2. DEFINITIONS

As used herein, the following terms shall have the following meanings:

"Alkyl:" refers to a saturated branched, straight chain or cyclichydrocarbon radical. Typical alkyl groups include, but are not limitedto, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl,isopentyl, hexyl, and the like. In preferred embodiments, the alkylgroups are (C₁ -C₆)alkyl.

"Alkenyl:" refers to an unsaturated branched, straight chain or cyclichydrocarbon radical having at least one carbon-carbon double bond. Theradical may be in either the cis or trans conformation about the doublebond(s). Typical alkenyl groups include, but are not limited to,ethenyl, propenyl, isopropenyl, butenyl, isobutenyl, tert-butenyl,pentenyl, hexenyl and the like. In preferred embodiments, the alkenylgroup is (C₁ -C₆) alkenyl.

"Alkynyl:" refers to an unsaturated branched, straight chain or cyclichydrocarbon radical having at least one carbon-carbon triple bond.Typical alkynyl groups include, but are not limited to, ethynyl,propynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like. Inpreferred embodiments, the alkynyl group is (C₁ -C₆) alkynyl.

"Aryl:" refers to an unsaturated cyclic hydrocarbon radical having aconjugated π electron system. Typical aryl groups include, but are notlimited to, penta-2,4-diene, phenyl, naphthyl, anthracyl, azulenyl,chrysenyl, coronenyl, fluoranthenyl, indacenyl, idenyl, ovalenyl,perylenyl, phenalenyl, phenanthrenyl, picenyl, pleiadenyl, pyrenyl,pyranthrenyl, rubicenyl, and the like. In preferred embodiments, thearyl group is (C₅ -C₂₀) aryl, with (C₅ -C₁₀) being particularlypreferred.

"Alkaryl:" refers to a straight-chain alkyl, alkenyl or alkynyl groupwherein one of the hydrogen atoms bonded to a terminal carbon isreplaced with an aryl moiety. Typical alkaryl groups include, but arenot limited to, benzyl, benzylidene, benzylidyne, benzenobenzyl,naphthenobenzyl and the like. In preferred embodiments, the alkarylgroup is (C₆ -C₂₆) alkaryl, i.e., the alkyl, alkenyl or alkynyl moietyof the alkaryl group is (C₁ -C₆) and the aryl moiety is (C₅ -C₂₀). Inparticularly preferred embodiments, the alkaryl group is (C₆ -C₁₃)alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl groupis (C₁ -C₃) and the aryl moiety is (C5-C₁₀).

"Heteroaryl:" refers to an aryl moiety wherein one or more carbon atomsis replaced with another atom, such as N, P, O, S, As, Se, Si, Te, etc.Typical heteroaryl groups include, but are not limited to, acridarsine,acridine, arsanthridine, arsindole, arsindoline, carbazole, β-carboline,chromene, cinnoline, furan, imidazole, indazole, indole, indolizine,isoarsindole, isoarsinoline, isobenzofuran, isochromene, isoindole,isophosphoindole, isophosphinoline, isoquinoline, isothiazole,isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline,phenazine, phosphoindole, phosphinoline, phthalazine, pteridine, purine,pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole,pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline,selenophene, tellurophene, thiophene and xanthene. In preferredembodiments, the heteroaryl group is a 5-20 membered heteroaryl, with5-10 membered aryl being particularly preferred.

"Alkheteroaryl:" refers to a straight-chain alkyl, alkenyl or alkynylgroup where one of the hydrogen atoms bonded to a terminal carbon atomis replaced with a heteroaryl moiety. In preferred embodiments, thealkheteroaryl group is 6-26 membered alkheteroaryl, i.e., the alkyl,alkenyl or alkynyl moiety of the alkheteroaryl is (C₁ -C₆) and theheteroaryl is a 5-20-membered heteroaryl. In particularly preferredembodiments the alkheteroaryl is 6-13 membered alkheteroaryl, i.e., thealkyl, alkenyl or alkynyl moiety is a 5-10 membered heteroaryl.

"Substituted Alkyl, Alkenyl, Alkynyl, Aryl Alkaryl, Heteroaryl orAlkheteroaryl:" refers to an alkyl, alkenyl, alkynyl, aryl, alkaryl,heteroaryl or alkheteroaryl group in which one or more hydrogen atoms isreplaced with another substituent. Preferred substituents include --OR,--SR, --NRR, --NO₂, --CN, halogen, --C(O)R, --C(O)OR and --C(O)NR, whereeach R is independently hydrogen, alkyl, alkenyl, alkynyl, aryl,alkaryl, heteroaryl or alkheteroaryl.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a Schiffer-Edmundson helical wheel diagram of an idealizedamphipathic α-helix in which open circles represent hydrophilic aminoacid residues and shaded circles represent hydrophobic amino acidresidues.

FIG. 1B is a helical net diagram of the idealized amphipathic helix ofFIG. 1A.

FIG. 1C is a helical cylinder diagram of the idealized amphipathic helixof FIG. 1A.

FIG. 2A is a Schiffer-Edmundson helical wheel diagram of the corepeptide of structure (I) illustrating the amphipathicity of the helix(open circles represent hydrophilic amino acid residues, shaded circlesrepresent hydrophobic amino acid residues and partially shaded circlesrepresent either hydrophilic or hydrophobic amino acid residues).

FIG. 2B is a helical net diagram of the core peptide of structure (I)illustrating the hydrophobic face of the helix.

FIG. 2C is a helical net diagram of the core peptide of structure (I)illustrating the hydrophilic face of the helix.

FIG. 3A is a helical net diagram illustrating the hydrophilic face ofSegrest's consensus 22-mer peptide (PVLDEFREKLNEELEALKQKLK; SEQ IDNO:75).

FIG. 3B is a helical net diagram illustrating the hydrophilic face ofexemplary core peptide 4 (PVLDLFRELLNELLEALKQKLK; SEQ ID NO:4).

FIG. 4A is a helical net diagram illustrating the hydrophobic face ofSegrest's consensus 22-mer peptide (SEQ ID NO:75).

FIG. 4B is a helical net diagram illustrating the hydrophobic face ofexemplary core peptide 4 (SEQ ID NO:4).

FIG. 5A is a Schiffer-Edmundson helical wheel diagram of Segrest'sconsensus 22-mer peptide (SEQ ID NO:75).

FIG. 5B is a Schiffer-Edmundson helical wheel diagram of exemplary corepeptide 4 (SEQ ID NO:4).

FIG. 6A is a computer model of two peptides 4 (SEQ ID NO:4) arranged inan antiparallel fashion in which residues Glu-8 and Gln-19 arehighlighted to illustrate the ability of these two peptides to formintermolecular hydrogen-bonds when bound to lipids.

FIG. 6B is a computer model of two peptides 102 (PVLDLFRELLNLXLEALKEKLK;SEQ ID NO:102) arranged in an antiparallel fashion in which residuesGlu-8 and Glu-19 are highlighted to illustrate the inability of thesetwo peptides to form intermolecular hydrogen-bonds when bound to lipids.

FIG. 7A illustrates a tertiary-order branched network of the invention.

FIG. 7B illustrates a quaternary-order branched network of theinvention.

FIG. 7C illustrates a mixed-order branched network of the invention.

FIG. 7D illustrates exemplary "Lys-tree" branched networks of theinvention.

FIG. 8A is a graph illustrating the differences between the observed Hαchemical shifts and the tabulated random coil Hα chemical shifts forpeptide 4 (SEQ ID NO:4) and Segrest's consensus 22-mer peptide (SEQ IDNO:75).

FIG. 8B is a graph illustrating the differences between the observedamide proton chemical shifts and the tabulated random coil amide protonchemical shifts for peptide 4 (SEQ ID NO:4) and Segrest's consensus22-mer peptide (SEQ ID NO:75).

FIG. 8C is a cartoon illustrating the periodic relationship between theHα proton chemical shifts of peptide 4 (SEQ ID NO:4) and its α-helicalconformation.

FIGS. 9A-9D provide gel chromatograms of isolated human HDL incubatedfor 2 hr. at 37° C. in buffer as measured by absorbance (--) and in thepresence of ¹⁴ C-labeled peptide 4 as measured by absorbance (- - -) or¹⁴ C-radiometric counting (♦). Chromatograms were obtained atpeptide:HDL mass ratios of 1:15 (FIG. 9A), 1:10 (FIG. 9B), 1:5 (FIG. 9C)and 1:3 (FIG. 9D).

FIG. 9E is a control gel filtration chromatogram of free, unbound ¹⁴C-labeled peptide 4 as measured by absorbance (- - -) and ¹⁴ Cradiometric counting (♦).

FIG. 9F provides gel filtration difference chromatograms illustratingthe difference between each of the peptide-treated HDL absorbancechromatograms presented in FIGS. 9A (- - -), 9B (--), 9C (- - -) and 9D(--) and the control chromatogram of FIG. 9E (positive values indicate ahigher absorbance in the treated sample; negative values indicate agreater absorbance in the control sample).

FIG. 10 is a graph illustrating the lipoprotein profile of rabbitsinjected with 10 mg/kg peptide 4 (SEQ ID NO: 4) (in the form ofpeptide/DPPC complexes).

FIG. 11A is a cartoon depicting the various aggregation states andpeptide-lipid complexes that can be obtained with the ApoA-I agonists ofthe invention. Left: Multimerization process of the peptides resultingfrom the interaction of several peptide helices and leading to theformation of oligomers in conditions of defined peptide concentration,pH and ionic strength. Center: The interaction of the peptides (in anyof these states of aggregation) with lipidic entities (such as SUVs)leads to lipid reorganization. Right: By changing the lipid:peptidemolar ratio, different types of peptide-lipid complexes can be obtained,from lipid-peptide comicelles at low lipid-peptide ratios, to discoidalparticles and finally to large multilamellar complexes at increasinglyhigher lipid:peptide ratios.

FIG. 11B illustrates the generally-accepted model for discoidalpeptide-lipid complexes formed in a defined range of lipid:peptideratios. Each peptide surrounding the disc edge is in close contact withits two nearest neighbors.

5. DETAILED DESCRIPTION OF THE INVENTION

The ApoA-I agonists of the invention mimic ApoA-I function and activity.They form amphipathic helices (in the presence of lipids), bind lipids,form pre-β-like or HDL-like complexes, activate LCAT, increase serum HDLconcentration and promote cholesterol efflux. The biological function ofthe peptides correlates with their helical structure, or conversion tohelical structures in the presence of lipids.

The ApoA-I agonists of the invention can be prepared in stable bulk orunit dosage forms, e.g., lyophilized products, that can be reconstitutedbefore use in vivo or reformulated. The invention includes thepharmaceutical formulations and the use of such preparations in thetreatment of hyperlipidemia, hypercholesterolemia, coronary heartdisease, atherosclerosis, and other conditions such as endotoxemiacausing septic shock.

The invention is illustrated by working examples which demonstrate thatthe ApoA-I agonists of the invention associate with the HDL component ofplasma, and can increase the concentration of HDL and pre-β particles.The ApoA-I agonists of the invention increase cellular cholesterolefflux. The agonists are also extremely efficient at activating LCAT,and thus promote RCT. Use of the ApoA-I agonists of the invention invivo in animal models results in an increase in serum HDL concentration.

The invention is set forth in more detail in the subsections below,which describe: the composition and structure of the ApoA-I peptideagonists; structural and functional characterization; methods ofpreparation of bulk and unit dosage formulations; and methods of use.

5.1. PEPTIDE STRUCTURE AND FUNCTION

The ApoA-I agonists of the invention are generally peptides, oranalogues thereof, which are capable of forming amphipathic α-helices inthe presence of lipids and which mimic the activity of ApoA-I. Theagonists have as their main feature a "core" peptide composed of 15 to29 amino acid residues, preferably 22 amino acid residues, or ananalogue thereof wherein at least one amide linkage in the peptide isreplaced with a substituted amide, an isostere of an amide or an amidemimetic.

The ApoA-I agonists of the invention are based, in part, on theapplicants' surprising discovery that altering certain amino acidresidues in the primary sequence of the 22-mer consensus sequence ofVenkatachalapathi et al., 1991, Mol. Conformation and Biol.Interactions, Indian Acad. Sci. B:585-596 (PVLDEFREKLNEELEALKQKLK; SEQID NO:75; hereinafter "Segrest's consensus 22-mer" or "consensus22-mer") that were thought to be critical for activity yields syntheticpeptides which exhibit activities that approach, or in some embodimentseven exceed, the activity of native ApoA-I. In particular, theapplicants have discovered that replacing three charged amino acidresidues in Segrest's consensus 22-mer peptide (Glu-5, Lys-9 and Glu-13)with a hydrophobic Leu residue provides peptides that mimic thestructural and functional properties of ApoA-I to a degree that isunprecedented in the art.

While not intending to be bound by any particular theory, it is believedthat the helix formed by the ApoA-I agonists of the invention moreclosely mimics the structural and functional properties of theamphipathic helical regions of native ApoA-I that are important foreffecting lipid-binding, cholesterol efflux and LCAT activation thandoes the α-helix formed by the ApoA-I mimetic peptides described in theliterature, thereby resulting in peptides that exhibit significantlyhigher ApoA-I-like activity than these other peptides. Indeed, whereasmany of the ApoA-I agonists of the invention approach, and in someembodiments even exceed, the activity of ApoA-I, to date, the bestpeptide ApoA-I mimics described in the literature--peptide 18AM4(EWLEAFYKKVLEKLKELF; SEQ ID NO: 246) (Corinjn et al., 1993, Biochim.Biophys. Acta 1170:8-16; Labeur et al., Oct. 1994, Arteriosclerosis:Abstract Nos. 186 and 187) and N-acetylated, C-amidated peptide 18AM4(SEQ ID NO: 239) (Brasseur, 1993, Biochim. Biophys. Acta1170:1-7)--exhibit less than 4% and 11%, respectively, of the activityof ApoA-I as measured by the LCAT activation assay described herein.

Generally, the core peptides (or analogues thereof) that compose theApoA-I agonists of the invention have the following structural formula(I):

    X.sub.1 -X.sub.2 -X.sub.3 -X.sub.4 -X.sub.5 -X.sub.6 -X.sub.7 -X.sub.8 -X.sub.9 -X.sub.10 -X.sub.11 -X.sub.12 -X.sub.13 -X.sub.14 -X.sub.15 -X.sub.16 -X.sub.17 -X.sub.18 -X.sub.19 -X.sub.20 -X.sub.21 -X.sub.22

wherein:

X₁ is Pro (P), Ala (A), Gly (G), Gln (Q), Asn (N), Asp (D) or D-Pro (p);

X₂ is an aliphatic amino acid;

X₃ is Leu (L) or Phe (F);

X₄ is an acidic amino acid;

X₅ is Leu (L) or Phe (F);

X₆ is Leu (L) or Phe (F);

X₇ is a hydrophilic amino acid;

X₈ is an acidic or a basic amino acid;

X₉ is Leu (L) or Gly (G);

X₁₀ is Leu (L), Trp (W) or Gly (G);

X₁₁ is a hydrophilic amino acid;

X₁₂ is a hydrophilic acid;

X₁₃ is Gly (G) or an aliphatic amino acid;

X₁₄ is Leu (L), Trp (W), Gly (G) or Nal;

X₁₅ is a hydrophilic amino acid;

X₁₆ is a hydrophobic amino acid;

X₁₇ is a hydrophobic amino acid;

X₁₈ is a basic amino acid, Gln (Q) or Asn (N);

X₁₉ is a basic amino acid, Gln (Q) or Asn (N);

X₂₀ is a basic amino acid;

X₂₁ is an aliphatic amino acid; and

X₂₂ is a basic amino acid.

The core peptides of structure (I) are defined, in part, in terms ofamino acids of designated classes. The definitions of the variousdesignated classes are provided infra in connection with the descriptionof mutated or altered embodiments of structure (I).

In the core peptides of structure (I), the symbol "--" between aminoacid residues X_(n) generally designates a backbone constitutive linkingfunction. Thus, the symbol "--" usually represents a peptide bond oramide linkage (--C(O)NH--). It is to be understood, however, that thepresent invention contemplates peptide analogues wherein one or moreamide linkages is optionally replaced with a linkage other than amide,preferably a substituted amide or an isostere of amide. Thus, while thevarious X_(n) residues within structure (I) are generally described interms of amino acids, and preferred embodiments of the invention areexemplified by way of peptides, one having skill in the art willrecognize that in embodiments having non-amide linkages, the term "aminoacid" or "residue" as used herein refers to other bifunctional moietiesbearing groups similar in structure to the side chains of the aminoacids.

Substituted amides generally include, but are not limited to, groups ofthe formula --C(O)NR--, where R is (C₁ -C₆) alkyl, substituted (C₁ -C₆)alkyl, (C₁ -C₆) alkenyl, substituted (C₁ -C₆) alkenyl, (C₁ -C₆) alkynyl,substituted (C₁ -C₆) alkynyl, (C₅ -C₂₀) aryl, substituted (C₅ -C₂₀)aryl, (C₆ -C₂₆) alkaryl, substituted (C₆ -C₂₆) alkaryl, 5-20 memberedheteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl and substituted 6-26 membered alkheteroaryl.

Isosteres of amide generally include, but are not limited to, --CH₂NH--, --CH₂ S--, --CH₂ CH₂ --, --CH═CH-- (cis and trans), --C(O)CH₂ --,--CH(OH)CH₂ -- and --CH₂ SO--. Compounds having such non-amide linkagesand methods for preparing such compounds are well-known in the art (see,e.g., Spatola, March 1983, Vega Data Vol. 1, Issue 3; Spatola, 1983,"Peptide Backbone Modifications" In: Chemistry and Biochemistry of AminoAcids Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p.267 (general review); Morley, 1980, Trends Pharm. Sci. 1:463-468; Hudsonet al., 1979, Int. J. Prot. Res. 14:177-185 (--CH₂ NH--, --CH₂ CH₂ --);Spatola et al., 1986, Life Sci. 38:1243-1249 (--CH₂ --S); Hann, 1982, J.Chem. Soc. Perkin Trans. I. 1:307-314 (--CH═CH--, cis and trans);Almquist et al., 1980, J. Med. Chem. 23:1392-1398 (--COCH₂ --);Jennings-White et al., Tetrahedron. Lett. 23:2533 (--COCH₂ --); EuropeanPatent Application EP 45665 (1982) CA 97:39405 (--CH(OH)CH₂ --);Holladay et al., 1983, Tetrahedron Lett. 24:4401-4404 (--C(OH)CH₂ --);and Hruby, 1982, Life Sci. 31:189-199 (--CH₂ --S--)

Additionally, one or more amide linkages can be replaced withpeptidomimetic or amide mimetic moieties which do not significantlyinterfere with the structure or activity of the peptides. Suitable amidemimetic moieties are described, for example, in Olson et al., 1993, J.Med. Chem. 36:3039-3049.

A critical feature of the core peptides of structure (I), is theirability to form an amphipathic α-helix in the presence of lipids. Byamphipathic is meant that the α-helix has opposing hydrophilic andhydrophobic faces oriented along its long axis, i.e., one face of thehelix projects mainly hydrophilic side chains while the opposite faceprojects mainly hydrophobic side chains. FIGS. 1A and 1B present twoillustrative views of the opposing hydrophilic and hydrophobic faces ofan exemplary idealized amphipathic α-helix. FIG. 1A is aSchiffer-Edmundson helical wheel diagram (Schiffer and Edmundson, 1967,Biophys. J. 7:121-135). In the wheel, the long axis of the helix isperpendicular to the page. Starting with the N-terminus, successiveamino acid residues (represented by circles) are radially distributedabout the perimeter of a circle at 100° intervals. Thus, amino acidresidue n+1 is positioned 100° from residue n, residue n+2 is positioned100° from residue n+1, and so forth. The 100° placement accounts for the3.6 amino acid residues per turn that are typically observed in anidealized α-helix. In FIG. 1A, the opposing hydrophilic and hydrophobicfaces of the helix are clearly visible; hydrophilic amino acids arerepresented as open circles and hydrophobic amino acid residues arerepresented as shaded circles.

FIG. 1B presents a helical net diagram of the idealized amphipathichelix of FIG. 1A. (Lim, 1978, FEBS Lett. 89:10-14). In a typical helicalnet diagram, the α-helix is presented as a cylinder that has been cutalong the center of its hydrophilic face and flattened. Thus, the centerof the hydrophobic face, determined by the hydrophobic moment of thehelix (Eisenberg et al., 1982, Nature 299:371-374), lies in the centerof the figure and is oriented so as to rise out of the plane of thepage. An illustration of the helical cylinder prior to being cut andflattened is depicted in FIG. 1C. By cutting the cylinder alongdifferent planes, different views of the same amphipathic helix can beobserved, and different information about the properties of the helixobtained.

The amphipathic nature of the α-helix formed by the core peptides ofstructure (I) in the presence of lipids is illustrated in FIG. 2. FIG.2A presents a Schiffer-Edmundson helical wheel diagram, FIG. 2B presentsa helical net diagram illustrating the hydrophobic face and FIG. 2Cpresents a helical net diagram illustrating the hydrophilic face. Ineach of FIGS. 2A, 2B and 2C, hydrophilic residues are represented asopen circles and hydrophobic residues as shaded circles. As will bediscussed more thoroughly below in conjunction with altered or mutatedforms of the peptides of structure (I), certain amino acid residues canbe replaced with other amino acid residues such that the hydrophilic andhydrophobic faces of the helix formed by the peptides may not becomposed entirely of hydrophilic and hydrophobic amino acids,respectively. Thus, it is to be understood that when referring to theamphipathic α-helix formed by the core peptides of the invention, thephrase "hydrophilic face" refers to a face of the helix having overallnet hydrophilic character. The phrase "hydrophobic face" refers to aface of the peptide having overall net hydrophobic character.

While not intending to be bound by any particular theory, it is believedthat certain structural and/or physical properties of the amphipathichelix formed by the core peptides of structure (I), are important foractivity. These properties include the degree of amphipathicity, overallhydrophobicity, mean hydrophobicity, hydrophobic and hydrophilic angles,hydrophobic moment, mean hydrophobic moment, and net charge of theα-helix.

While the helical wheel diagrams of FIG. 2A provide a convenient meansof visualizing the amphipathic nature of the core peptides of structure(I), the degree of amphipathicity (degree of asymmetry ofhydrophobicity) can be conveniently quantified by calculating thehydrophobic moment (μ_(H)) of the helix. Methods for calculating μ_(H)for a particular peptide sequence are well-known in the art, and aredescribed, for example in Eisenberg, 1984, Ann. Rev. Biochem.53:595-623. The actual μ_(H) obtained for a particular peptide willdepend on the total number of amino acid residues composing the peptide.Thus, it is generally not informative to directly compare μ_(H) forpeptides of different lengths.

The amphipathicities of peptides of different lengths can be directlycompared by way of the mean hydrophobic moment (<μ_(H) >). The meanhydrophobic moment can be obtained by dividing μ_(H) by the number ofresidues in the helix (i.e., <μ_(H) >=μ_(H) /N). Generally, corepeptides which exhibit a <μ_(H) > in the range of 0.45 to 0.65, asdetermined using the normalized consensus hydrophobicity scale ofEisenberg (Eisenberg, 1984, J. Mol. Biol. 179:125-142) are considered tobe within the scope of the present invention, with a <μ_(H) > in therange of 0.50 to 0.60 being preferred.

The overall or total hydrophobicity (H_(o)) of a peptide can beconveniently calculated by taking the algebraic sum of thehydrophobicities of each amino acid residue in the peptide (i.e., H_(o)=##EQU1## H_(i)), where N is the number of amino acid residues in thepeptide and H_(i) is the hydrophobicity of the ith amino acid residue).The mean hydrophobicity (<H_(o) >) is the hydrophobicity divided by thenumber of amino acid residues (i.e., <H_(o) >=H_(o) /N). Generally, corepeptides that exhibit a mean hydrophobicity in the range of -0.050 to-0.070, as determined using the normalized consensus hydrophobicityscale of Eisenberg (Eisenberg, 1984, J. Mol. Biol. 179:125-142) areconsidered to be within the scope of the present invention, with a meanhydrophobicity in the range of -0.030 to -0.055 being preferred.

The total hydrophobicity of the hydrophobic face (H_(o) ^(pho)) of anamphipathic helix can be obtained by taking the sum of thehydrophobicities of the hydrophobic amino acid residues which fall intothe hydrophobic angle as defined below (i.e., H_(o) ^(pho) = ##EQU2##H_(i), where H_(i) is as previously defined and N_(H) is the totalnumber of hydrophobic amino acids in the hydrophobic face). The meanhydrophobicity of the hydrophobic face (<H_(o) ^(pho) >) is H_(o) ^(pho)/N_(H) where N_(H) is as defined above. Generally, core peptides whichexhibit a <H_(o) ^(pho) > in the range of 0.90 to 1.20, as determinedusing the consensus hydrophobicity scale of Eisenberg (Eisenberg, 1984,supra; Eisenberg et al., 1982, supra) are considered to be within thescope of the present invention, with a <H_(o) ^(pho) > in the range of0.94 to 1.10 being preferred.

The hydrophobic angle (pho angle) is generally defined as the angle orarc covered by the longest continuous stretch of hydrophobic amino acidresidues when the peptide is arranged in the Schiffer-Edmundson helicalwheel representation (i.e., the number of contiguous hydrophobicresidues on the wheel multiplied by 20°). The hydrophilic angle (phiangle) is the difference between 360° and the pho angle (i.e., 360°-phoangle). Those of skill in the art will recognize that the pho and phiangles will depend, in part, on the number of amino acid residues in thepeptide. For example, referring to FIGS. 5A and 5B, it can be seen thatonly 18 amino acids fit around one rotation of the Schiffer-Edmundsonhelical wheel. Fewer amino acids leave a gap in the wheel; more aminoacids cause certain positions of the wheel to be occupied by more thanone amino acid residue.

In the case of peptides containing more than 18 amino acid residues,such as the core peptides of structure (I), a "continuous" stretch ofhydrophobic amino acid residues is meant that at least one amino acid atpositions along the wheel containing two or more amino acids is ahydrophobic amino acid. Thus, referring to FIG. 5B, the pho angle is thearc covered by residues 5, 16, 9, 2, 13, 6, 17, 10, 3 and 14 despite theoccurrence of a hydrophilic residue at position 20, as the residue atposition 2, which shares the same position on the wheel, is ahydrophobic residue. Typically, core peptides having a pho angle in therange of 160° to 220° are considered to be within the scope of theinvention, with a pho angle in the range of 180° to 200° beingpreferred.

Certain structural and/or physical characteristics of the core peptidesof structure (I) are illustrated in FIGS. 3 and 4. FIG. 3B presents ahelical net diagram of an exemplary core peptide of the invention,peptide 4 (PVLDLFRELLNELLEALKQKLK; SEQ ID NO:4), illustrating the chargedistribution along the hydrophilic face of the helix. In FIG. 3B, thehelical cylinder has been cut along the center of the hydrophobic faceand flattened. The three hydrophobic Leu (L) residues that replacehydrophilic residues in Segrest's consensus 22-mer (FIG. 3A) are shaded.As can be seen in FIG. 3B, positively-charged amino acid residues areclustered at the last C-terminal turn of the helix (the C-terminus is atthe top of the page). While not intending to be bound by any particulartheory, it is believed that the cluster of basic residues at theC-terminus stabilizes the helix through charge (NH₃ ⁺)-helix dipoleelectrostatic interactions. It is also thought that stabilization occursthrough hydrophobic interactions between lysine side chains and thehelix core (see, Groebke et al., 1996, Proc. Natl. Acad. Sci. U.S.A.93:4025-4029; Esposito et al., 1997, Biopolymers 41:27-35).

With the exception of the positively-charged C-terminal cluster,negative charges are distributed on the rest of the hydrophilic face,with at least one negatively charged (acidic) amino acid residue perturn, resulting in a continuous stretch of negative charges along thehydrophilic face of the helix. One positive charge is located at residue7, which potentially contributes to helix stability by forming a saltbridge with an acidic residue one turn away on the helix.

FIG. 4B presents a helical net diagram illustrating the hydrophobic faceof the amphipathic helix formed by exemplary core peptide 4 (SEQ IDNO:4). In FIG. 4B, the helical cylinder is cut along the center of thehydrophilic face and flattened. The hydrophobic face of the core peptideconsists of two hydrophobic residues per turn, except for the lastC-terminal turn, where basic residues dominate. NMR studies indicatethat amino acid residues 3, 6, 9 and 10 of this core peptide form ahydrophobic cluster near the N-terminus of the helix. Phe-6 is centeredin this cluster and is believed to play an important role in stabilizingthe hydrophobic cluster.

While not intending to be bound by any particular theory, it is believedthat the hydrophobic cluster formed by residues 3, 6, 9 and 10 issignificant in effecting lipid binding and LCAT activation. For example,whereas exemplary peptide 4 (SEQ ID NO:4) exhibits 93% LCAT activationin the assay described herein, a derivative of peptide 4 containing aLys (K) residue at position 9 (peptide 33; SEQ ID NO:33), which destroysthe hydrophobic cluster, exhibits only 33% LCAT activation in the sameassay. Amphipathic peptides are expected to bind phospholipids bypointing their hydrophobic faces towards the alkyl chains of the lipidmoieties. Thus, it is believed that this highly hydrophobic clustercontributes to the strong lipid affinities observed for the corepeptides of the invention. Since lipid binding is a prerequisite forLCAT activation, it is believed that this hydrophobic cluster is alsoessential for LCAT activation.

Aromatic residues are often found to be important in anchoring peptidesand proteins to lipids (De Kruijff, 1990, Biosci. Rep. 10:127-130;O'Neil and De Grado, 1990, Science 250:645-651; Blondelle et al., 1993,Biochim. Biophys. Acta 1202:331-336). Thus, it is further believed thatPhe-6, which is positioned at the center of the hydrophobic cluster, mayalso play a key role in anchoring the core peptides of structure (I) tolipids.

Interactions between the core peptides of the invention and lipids leadto the formation of peptide-lipid complexes. As illustrated in FIG. 11A,the type of complex obtained (comicelles, discs, vesicles ormultilayers) depends on the lipid:peptide molar ratio, with comicellesgenerally being formed at low lipid:peptide molar ratios and discoidaland vesicular or multilayer complexes being formed with increasinglipid:peptide molar ratios. This characteristic has been described foramphipathic peptides (Epand, The Amphipathic Helix, 1993) and for ApoA-I(Jones, 1992, Structure and Function of Apolipoproteins, Chapter 8, pp.217-250). The lipid:peptide molar ratio also determines the size andcomposition of the complexes (see, Section 5.3.1, infra).

The long axis of the α-helix formed by the core peptides of structure(I) has an overall curved shape. In typical amphipathic helices, it hasbeen found that the lengths of the hydrogen bonds of the hydrophilic andhydrophobic faces vary such that the hydrophobic side of the helix isconcave (Barlow and Thornton, 1988, J. Mol. Biol. 201:601-619; Zhou etal., 1992, J. Am. Chem. Soc. 33:11174-11183; Gesell et al., 1997, J.Biomol. NMR 9:127-135). While not intending to be bound by theory, it isbelieved that the overall curvature of the hydrophobic face of the helixmay be important in binding discoidal complexes--a curved helix permitsthe peptide to "fit" better around the edges of discoidal particles,thereby increasing the stability of the peptide-disc complex.

In the generally accepted structural model of ApoA-I, the amphipathicα-helices are packed around the edge of the discoidal HDL (see, FIG.11B). In this model, the helices are assumed to be aligned with theirhydrophobic faces pointing towards the lipid acyl chains (Brasseur etal., 1990, Biochim. Biophys. Acta 1043:245-252). The helices arearranged in an antiparallel fashion, and a cooperative effect betweenthe helices is thought to contribute to the stability of the discoidalHDL complex (Brasseur et al., supra). It has been proposed that onefactor that contributes to the stability of the HDL discoidal complex isthe existence of ionic interactions between acidic and basic residuesresulting in the formation of intermolecular salt bridges or hydrogenbonds between residues on adjacent anti-parallel helices. In this model,the peptides are considered not as a single entity, but as ininteraction with at least two other neighboring peptide molecules (FIG.11B).

It is also generally accepted that intramolecular hydrogen bond or saltbridge formation between acidic and basic residues, respectively, atpositions i and i+3 of the helix stabilize the helical structure(Marqusee et al:, 1985, Proc. Natl. Acad. Sci. USA 84(24):8898-8902).

Thus, additional key features of the core peptides of structure (I) aretheir ability to form intermolecular hydrogen-bonds with one anotherwhen aligned in an antiparallel fashion with their hydrophobic facespointing in the same direction, such as would be the case when thepeptides are bound to lipids (i.e., between the acidic residues atpositions 4 and 8 and the basic residues at positions 18, 20 and 22),and also their ability to form intramolecular hydrogen bonds or saltbridges near the N- and C-termini of the helix (i.e., between the acidicand basic residues at positions 4 and 7 and 15 and 18).

The ability of the core peptides of structure (I) to form intermolecularhydrogen bonds is illustrated in FIG. 6A. In FIG. 6A, two idealα-helices of exemplary core peptide 4 (SEQ ID NO:4) are aligned in anantiparallel fashion with their respective hydrophobic faces pointing inthe same direction (out of the plane of the page). H-bondinginteractions could occur between residues E-8 and Q-19(Huyghues-Despointes et al., 1995, Biochemistry 34(41):13267-13271).

Furthermore, when arranged in this anti-parallel fashion, the helicesare closely packed; there is no steric hindrance preventing closecontact between the helices. Alterations in the sequence of the corepeptides which affect the packing of the helices negatively influencesthe activity of the core peptides. For example, referring to FIG. 6B, adimer of peptides having Gln-19 replaced by Glu-19 (peptide 102;PVLDLFRELLNLXLEALKEKLK where X is Aib; SEQ ID NO:102), and whichtherefore cannot form intermolecular hydrogen bonds, did not activateLCAT. Significantly, whereas peptide 4 (SEQ ID NO:4) exhibited 93% LCATactivation in the assay described herein, peptide 102 (SEQ ID NO:102)exhibited only 2% activity in the same assay.

Thus, while not being bound by any particular theory, it is believedthat the ability of the core peptides of structure (I) to closely packand ionically interact to form intra- and/or inter-molecular saltbridges and/or hydrogen bonds when bound to lipids in an antiparallelfashion is an important feature of the core peptides of the invention.

The ability of the core peptides to form favorable intermolecularpeptide-peptide interactions is also thought to be of relevance in theabsence of lipids. The core peptides of the invention self-associate,due in part to their high <μ_(H) >, <H₀ > and hydrophobic angle (see,TABLE I, infra). The self-association phenomenon depends on theconditions of pH, peptide concentration and ionic strength, and canresult in several states of association, from monomeric to severalmultimeric forms (FIG. 11A). The hydrophobic core of peptide aggregatesfavors hydrophobic interactions with lipids. The ability of the peptidesto aggregate even at very low concentrations may favor their binding tolipids. It is thought that in the core of the peptide aggregatespeptide-peptide interactions also occur and may compete withlipid-peptide interactions.

In addition to the above-described properties, other parameters arethought to be important for activity as well, including the total numberof hydrophobic residues, the total number of charged residues, and thenet charge of the peptides.

A summary of the preferred physical and structural properties of thecore peptides of structure (I) is provided in TABLE I, below:

                  TABLE I                                                         ______________________________________                                        PHYSICAL PROPERTIES OF PREFERRED                                                ApoA-I AGONISTS OF STRUCTURE (I)                                              PROPERTY       RANGE        PREFERRED RANGE                                 ______________________________________                                        % hydrophobic amino                                                                        40-70        50-60                                                 acids                                                                         <H.sub.o > -0.050 to -0.070 -0.030 to -0.055                                  <H.sub.o .sup.pho > 0.90-1.2  0.94-1.1                                        <μ.sub.H > 0.45-0.65 0.50-0.60                                             pho angle 1600-2200 1800-2000                                                 # positively 3-5 4                                                            charged amino acids                                                           # negatively 3-5 4                                                            charged amino acids                                                           net charge -1 to +1 0                                                       hydrophobic cluster                                                                        positions 3,6,9,10 are hydrophobic amino                            acids                                                                        acidic cluster at least 1 acidic amino acid per turn except                    for last 5 C-terminal amino acids                                            basic cluster at least 3 basic amino acids in last 5 C-                        terminal amino acids                                                       ______________________________________                                    

The properties of the amphipathic α-helices formed by the core peptidesof the invention differ significantly from the properties of class Aamphipathic α-helices, particularly the class A α-helix of Segrest'sconsensus 22-mer. These differences are illustrated with exemplary corepeptide 4 (SEQ ID NO:4) in FIGS. 3-5.

Referring to FIGS. 4A and 4B, it can be seen that the hydrophobic faceof peptide 4 has much greater hydrophobic character than the hydrophobicface of Segrest's consensus 22-mer. In particular, residue 5, 9 and 13(shaded region of FIG. 4B) are hydrophobic Leu (L) residues in peptide 4(SEQ ID NO:4) as compared to charged residues in the consensus 22-mer(SEQ ID NO:75). The replacement of these three charged residues inSegrest's consensus 22-mer with hydrophobic Leu (L) residues leads tosignificant differences in the amphipathicity, hydrophobicity, pho angleand other properties of the helix.

A comparison of the physical and structural properties of two exemplarycore peptides of structure (I), peptide 4 (SEQ ID NO:4) and peptide 8(SEQ ID NO:8), and Segrest's consensus 22-mer (SEQ ID NO:75) is providedin TABLE II, below:

                  TABLE II                                                        ______________________________________                                        COMPARISON OF PROPERTIES OF EXEMPLARY CORE                                      PEPTIDES WITH SEGREST'S CONSENSUS 22-MER                                        PROPERTY   CONSENSUS    peptide 4                                                                            peptide 8                                  ______________________________________                                        # amino acids                                                                            22           22       22                                             # hydrophilic 13 10 10                                                        amino acids                                                                   # hydrophobic 9 12 12                                                         amino acids                                                                   % hydrophobic 41 55 55                                                        amino acids                                                                   <H.sub.o > -0.293 -0.013 -0.040                                               <H.sub.o .sup.pho > 0.960 0.990 0.940                                         <μ.sub.H > 0.425 0.547 0.521                                               pho angle 100° 200° 200°                                 # positively 5 4 4                                                            charged amino                                                                 acids                                                                         # negatively 6 4 4                                                            charged amino                                                                 acids                                                                         net charge -1 0 0                                                           ______________________________________                                    

Most notably, the core peptides of structure (I) are composed of alarger percentage of hydrophobic residues, have a significantly larger<H_(o) > and <μ_(H) >, and have a two-fold larger pho angle (see FIGS.5A and 5B). These differences in properties lead to significantdifferences in activity. Whereas Segrest's consensus 22-mer (SEQ IDNO:75) exhibits only 10% LCAT activation as compared with native ApoA-Iin the assays described herein, peptides 4 (SEQ ID NO:4) and 8 (SEQ IDNO:8) exhibit 93% and 83% LCAT activation, respectively, as comparedwith native ApoA-I in the same assays. Peptide 1(PVLDLFRELLNELLEZLKQKLK; SEQ ID NO:1) and peptide 2(GVLDLFRELLNELLEALKQKLKK; SEQ ID NO:2), exhibited 120% and 105% LCATactivation, respectively, as compared with native ApoA-I in the sameassays.

Certain amino acid residues in the core peptides of structure (I) can bereplaced with other amino acid residues without significantlydeleteriously affecting, and in many cases even enhancing, the activityof the peptides. Thus, also contemplated by the present invention arealtered or mutated forms of the core peptides of structure (I) whereinat least one defined amino acid residue in the structure is substitutedwith another amino acid residue. As one of the critical featuresaffecting the activity of the core peptides of the invention is believedto be their ability to form α-helices in the presence of lipids thatexhibit the amphipathic and other properties described above, it will berecognized that in preferred embodiments of the invention, the aminoacid substitutions are conservative, i.e., the replacing amino acidresidue has physical and chemical properties that are similar to theamino acid residue being replaced.

For purposes of determining conservative amino acid substitutions, theamino acids can be conveniently classified into two maincategories--hydrophilic and hydrophobic--depending primarily on thephysical-chemical characteristics of the amino acid side chain. Thesetwo main categories can be further classified into subcategories thatmore distinctly define the characteristics of the amino acid sidechains. For example, the class of hydrophilic amino acids can be furthersubdivided into acidic, basic and polar amino acids. The class ofhydrophobic amino acids can be further subdivided into apolar andaromatic amino acids. The definitions of the various categories of aminoacids that define structure (I) are as follows:

"Hydrophilic Amino Acid" refers to an amino acid exhibiting ahydrophobicity of less than zero according to the normalized consensushydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophilic amino acids include Thr(T), Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) andArg (R).

"Acidic Amino Acid" refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

"Basic Amino Acid" refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg (R) and Lys (K).

"Polar Amino Acid" refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Asn (N), Gln (Q) Ser (S) and Thr (T).

"Hydrophobic Amino Acid" refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg, 1984, J. Mol. Biol.179:125-142. Genetically encoded hydrophobic amino acids include Pro(P), Ile (I), Phe (F), Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly(G) and Tyr (Y).

"Aromatic Amino Acid" refers to a hydrophobic amino acid with a sidechain having at least one aromatic or heteroaromatic ring. The aromaticor heteroaromatic ring may contain one or more substituents such as--OH, --SH, --CN, --F, --Cl, --Br, --I, --NO₂, --NO, --NH₂, --NHR,--NRR, --C(O)R, --C(O)OH, --C(O)OR, --C(O)NH₂, --C(O)NHR, --C(O)NRR andthe like where each R is independently (C₁ -C₆) alkyl, substituted (C₁-C₆) alkyl, (C₁ -C₆) alkenyl, substituted (C₁ -C₆) alkenyl, (C₁ -C₆)alkynyl, substituted (C₁ -C₆) alkynyl, (C₅ -C₂₀) aryl, substituted (C₅-C₂₀) aryl, (C₆ -C₂₆) alkaryl, substituted (C₆ -C₂₆) alkaryl, 5-20membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 memberedalkheteroaryl or substituted 6-26 membered alkheteroaryl. Geneticallyencoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).

"Nonpolar Amino Acid" refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded apolar amino acids include Leu (L), Val (V), Ile(I), Met (M), Gly (G) and Ala (A).

"Aliphatic Amino Acid" refers to a hydrophobic amino acid having analiphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala (A), Val (V), Leu (L) and Ile (I).

The amino acid residue Cys (C) is unusual in that it can form disulfidebridges with other Cys (C) residues or other sulfanyl-containing aminoacids. The ability of Cys (C) residues (and other amino acids with --SHcontaining side chains) to exist in a peptide in either the reduced free--SH or oxidized disulfide-bridged form affects whether Cys (C) residuescontribute net hydrophobic or hydrophilic character to a peptide. WhileCys (C) exhibits a hydrophobicity of 0.29 according to the normalizedconsensus scale of Eisenberg (Eisenberg, 1984, supra), it is to beunderstood that for purposes of the present invention Cys (C) iscategorized as a polar hydrophilic amino acid, notwithstanding thegeneral classifications defined above.

As will be appreciated by those of skill in the art, the above-definedcategories are not mutually exclusive. Thus, amino acids having sidechains exhibiting two or more physical-chemical properties can beincluded in multiple categories. For example, amino acid side chainshaving aromatic moieties that are further substituted with polarsubstituents, such as Tyr (Y), may exhibit both aromatic hydrophobicproperties and polar or hydrophilic properties, and can therefore beincluded in both the aromatic and polar categories. The appropriatecategorization of any amino acid will be apparent to those of skill inthe art, especially in light of the detailed disclosure provided herein.

Certain amino acid residues, called "helix breaking" amino acids, have apropensity to disrupt the structure of α-helices when contained atinternal positions within the helix. Amino acid residues exhibiting suchhelix-breaking properties are well-known in the art (see, e.g., Chou andFasman, Ann. Rev. Biochem. 47:251-276) and include Pro (P), Gly (G) andpotentially all D-amino acids (when contained in an L-peptide;conversely, L-amino acids disrupt helical structure when contained in aD-peptide). While these helix-breaking amino acid residues fall into thecategories defined above, with the exception of Gly (G) (discussedinfra), these residues should not be used to substitute amino acidresidues at internal positions within the helix--they should only beused to substitute 1-3 amino acid residues at the N-terminus and/orC-terminus of the peptide.

While the above-defined categories have been exemplified in terms of thegenetically encoded amino acids, the amino acid substitutions need notbe, and in certain embodiments preferably are not, restricted to thegenetically encoded amino acids. Indeed, many of the preferred peptidesof structure (I) contain genetically non-encoded amino acids. Thus, inaddition to the naturally occurring genetically encoded amino acids,amino acid residues in the core peptides of structure (I) may besubstituted with naturally occurring non-encoded amino acids andsynthetic amino acids.

Certain commonly encountered amino acids which provide usefulsubstitutions for the core peptides of structure (I) include, but arenot limited to, β-alanine (β-Ala) and other omega-amino acids such as3-aminopropionic acid, 2,3-diaminopropionic acid (Dpr), 4-aminobutyricacid and so forth; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid(Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly);ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA);t-butylglycine (t-BuG); N-methylisoleucine (MeIle); phenylglycine (Phg);cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal);4-chlorophenylalanine (Phe(4-Cl)); 2-fluorophenylalanine (Phe(2-F));3-fluorophenylalanine (Phe(3-F)); 4-fluorophenylalanine (Phe(4-F));penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MSO);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); p-aminophenylalanine (Phe (pNH₂));N-methyl valine (MeVal); homocysteine (hCys), homophenylalanine (hPhe)and homoserine (hSer); hydroxyproline (Hyp), homoproline (hPro),N-methylated amino acids and peptoids (N-substituted glycines).

The classifications of the genetically encoded and common non-encodedamino acids according to the categories defined above are summarized inTABLE III, below. It is to be understood that TABLE III is forillustrative purposes only and does not purport to be an exhaustive listof amino acid residues that can be used to substitute the core peptidesdescribed herein. Other amino acid residues not specifically mentionedherein can be readily categorized based on their observed physical andchemical properties in light of the definitions provided herein.

                  TABLE III                                                       ______________________________________                                        CLASSIFICATIONS OF COMMONLY ENCOUNTERED                                         AMINO ACIDS                                                                               Genetically  Non-Genetically                                      Classification Encoded Encoded                                              ______________________________________                                        Hydrophobic                                                                     Aromatic F, Y, W Phg, Nal, Thi, Tic, Phe(4-                                     Cl), Phe(2-F), Phe(3-F),                                                      Phe(4-F), hPhe                                                              Apolar L, V, I, M, G, A, P t-BuA, t-BuG, MeIle, Nle,                            MeVal, Cha, McGly, Aib                                                      Aliphatic A, V, L, I b-Ala, Dpr, Aib, Aha, MeGly,                               t-BuA, t-BuG, MeIle, Cha,                                                     Nle, MeVal                                                                  Hydrophilic                                                                   Acidic D, E                                                                   Basic H, K, R Dpr, Orn, hArg, Phe(p-NH.sub.2),                                  Dbu, Dab                                                                    Polar C, Q, N, S, T Cit, AcLys, MSO, bAla, hSer                               Helix-Breaking P, G D-Pro and other D-amino                                     acids (in L-peptides)                                                     ______________________________________                                    

While in most instances, the amino acids of the core peptides ofstructure (I) will be substituted with L-enantiomeric amino acids, thesubstitutions are not limited to L-enantiomeric amino acids. Thus, alsoincluded in the definition of "mutated" or "altered" forms are thosesituations where an L-amino acid is replaced with an identical D-aminoacid (e.g., L-Arg→D-Arg) or with a D-amino acid of the same category orsubcategory (e.g., L-Arg→D-Lys), and vice versa. Indeed, in certainpreferred embodiments that are suitable for oral administration toanimal subjects, the peptides may advantageously be composed of at leastone D-enantiomeric amino acid. Peptides containing such D-amino acidsare thought to be more stable to degradation in the oral cavity, gut orserum than are peptides composed exclusively of L-amino acids.

As noted above, D-amino acids tend to disrupt the structure of α-heliceswhen contained at internal positions with an α-helical L-peptide.Furthermore, it has been observed that certain mutated forms of the corepeptides of structure (I) that are composed entirely of D-amino acidsexhibit significantly lower LCAT activation in the assay describedherein than identical peptides composed entirely of L-amino acids. As aconsequence, D-amino acids should not be used to substitute internalL-amino acids; D-amino acid substitutions should be limited to 1-3 aminoacid residues at the N-terminus and/or C-terminus of the peptide.

As previously discussed, the amino acid Gly (G) generally acts as ahelix-breaking residue when contained at internal positions of apeptide. Quite surprisingly, the applicants have discovered that whilethe helical structure of the core peptides of the invention is disruptedin the absence of lipids when internal amino acid residues aresubstituted with Gly (G), in the presence of lipids such Gly (G)containing peptides exhibit significant helical structure, as well asactivity. For example, whereas peptide 8 (SEQ ID NO:8) exhibits only 20%helical structure in buffer, 61-93% helical structure was observed inthe presence of lipids and 93% helicity was observed in the presence oftri-fluoroethanol (TFE). The helical structure of this peptide in thepresence of TFE was confirmed via NMR (see, Section 7.3.5, infra).Notably, this peptide also exhibited 83% LCAT activation. Other corepeptides containing internal glycine residues also exhibited ≧38% LCATactivation (see, e.g., TABLE X, Section 8.3, infra). Thus, although Gly(G) is generally considered to be a helix-breaking residue, Gly (G) canbe used to substitute amino acids at internal positions of the corepeptides of structure (I). Preferably, only internal residues positionedwithin about ±1 helical turn of the center of the peptide (particularlyfor peptides composed of an even number of amino acids) are substitutedwith Gly (G). Additionally, it is preferred that only one internal aminoacid residue in the peptide be substituted with Gly (G). Preferredembodiments of the ApoA-I agonists of the invention containing internalglycines are described in Section 5.1.2, infra.

Using the amino acid residue classifications described above inconjunction with the Schiffer-Edmundson helical wheel and helical netdiagram presentations of the core peptides of structure (I), as well asthe detailed description of the desired properties provided herein,altered or mutated forms of the core peptides of structure (I) thatsubstantially retain the amphipathic and other properties of the helix,and which are therefore considered to be within the scope of the presentinvention, can be readily obtained.

In a preferred embodiment of the invention, altered or mutated forms ofthe core peptides of structure (I) are obtained by fixing the thehydrophilic or hydrophobic residues according to structure (I) andsubstituting at least one non-fixed residue with another amino acid,preferably with another amino acid of the same category or sub-category.The residues composing the basic and/or hydrophobic clusters can also befixed, and at least one non-fixed residue substituted.

In another preferred embodiment, altered or mutated forms of the corepeptides of structure (I) are obtained by fixing the hydrophilic aminoacid residues positioned within the hydrophilic face of the helixaccording to structure (I) and substituting at least one non-fixed aminoacid residue with another amino acid, preferably with another amino acidresidue of the same category or sub-category. Referring to FIG. 2A, itcan be seen that residues 1, 4, 7, 8, 11, 12, 15, 18, 19 and 22 arepositioned within the hydrophilic face of the amphipathic helix formedby the core peptides of structure (I). Of these residues, all arehydrophilic except for residue 1, which may be either hydrophilic orhydrophobic. Thus, in one preferred embodiment, residues 4, 7, 8, 11,12, 15, 18, 19 and 22 are fixed according to structure (I) and at leastone of residues 2, 3, 5, 6, 9, 10, 13, 14, 16, 17, 20 and 21 issubstituted with another amino acid of the same category, preferablywith another amino acid of the same sub-category. Alternatively, residue1 is also fixed according to structure (I) and at least one of residues2, 3, 5, 6, 9, 10, 13, 14, 16, 17, 20 and 21 is substituted asdescribed.

In a particularly preferred embodiment, the C-terminal basic cluster(residues 18, 19, 20 and 22) is also fixed according to structure (I),and only residues 2, 3, 5, 6, 9, 10, 13, 14, 16, 17 and/or 21 aresubstituted.

In another particularly preferred embodiment, the hydrophobic cluster isalso fixed, and only residues 2, 5, 13, 14, 16, 17, 20 and/or 21 aresubstituted.

In still another particularly preferred embodiment, both the basic andhydrophobic clusters are fixed and only residues 2, 5, 13, 14, 16, 17and/or 21 are substituted.

In another preferred embodiment of the invention, altered or mutatedforms of the core peptides of the invention are obtained by fixing thehydrophobic amino acid residues positioned within the hydrophobic faceof the helix and substituting at least one non-fixed amino acid residuewith another amino acid residue, preferably with another residue of thesame category or sub-category.

Referring to FIG. 2A, it can be seen that residues 2, 3, 5, 6, 9, 10,13, 14, 16, 17, 20 and 21 are positioned within the hydrophobic face. Ofthese, all are hydrophobic except for residue 20, which is hydrophilic.Thus, in one preferred embodiment residues 2, 3, 5, 6, 9, 10, 13, 14,16, 17 and 21 are fixed according to structure (I) and at least one ofresidues 1, 4, 7, 8, 11, 12, 15, 18, 19, 20 and 22 is substituted withanother amino acid residue, preferably with another amino acid of thesame category or subcategory.

In a particularly preferred embodiment, the C-terminal basic cluster isalso fixed, and only residues 1, 4, 7, 8, 11, 12 and/or 15 aresubstituted.

In another embodiment, altered or mutated forms of the peptides ofstructure (I) are obtained by fixing all of the amino acid residuesresiding within the hydrophobic or hydrophilic face of the helix andsubstituting, preferably conservatively, at least one amino acid residueresiding in the other face with another amino acid residue. The residuescomprising the hydrophobic cluster and/or the basic cluster may also beoptionally fixed according to structure (I), as previously defined.

In another embodiment of the invention, the altered or mutated forms ofstructure (I) are obtained by substituting at least one amino acid witha non-conservative amino acid. Those of skill in the art will recognizethat such substitutions should not substantially alter the amphipathicand/or structural properties of the helix discussed, supra. Thus, incertain instances it may be desirable to substitute one or more pairs ofamino acids so as to preserve the net properties of the helix. Furtherguidance for selecting appropriate amino acid substitutions is providedby the peptide sequences listed in TABLE X (see, Section 8.3, infra).

In still another embodiment of the invention, the first one to fouramino acid residues at the N-terminus and/or C-terminus of the corepeptides of structure (I) are substituted with one or more amino acidresidues, or one or more peptide segments, that are known to conferstability to regions of α-helical secondary structure ("end-cap"residues or segments). Such end-cap residues and segments are well-knownin the art (see, e.g., Richardson and Richardson, 1988, Science240:1648-1652; Harper et al., 1993, Biochemistry 32(30):7605-7609;Dasgupta and Bell, 1993, Int. J. Peptide Protein Res. 41:499-511; Sealeet al., 1994, Protein Science 3:1741-1745; Doig et al., 1994,Biochemistry 33:3396-3403; Zhou et al., 1994, Proteins 18:1-7; Doig andBaldwin, 1995, Protein Science 4:1325-1336; Odaert et al., 1995,Biochemistry 34:12820-12829; Petrukhov et al., 1996, Biochemistry35:387-397; Doig et al., 1997, Protein Science 6:147-155).Alternatively, the first one to four N-terminal and/or C-terminal aminoacid residues of structure (I) can be replaced with peptidomimeticmoieties that mimic the structure and/or properties of end-cap residuesor segments. Suitable end-cap mimetics are well-known in the art, andare described, for example, in Richardson and Richardson, 1988, Science240:1648-1652; Harper et al., 1993, Biochemistry 32(30):7605-7609;Dasgupta and Bell, 1993, Int. J. Peptide Protein Res. 41:499-511; Sealeet al., 1994, Protein Science 3:1741-1745; Doig et al., 1994,Biochemistry 33:3396-3403; Zhou et al., 1994, Proteins 18:1-7; Doig andBaldwin, 1995, Protein Science 4:1325-1336; Odaert et al., 1995,Biochemistry 34:12820-12829; Petrukhov et al., 1996, Biochemistry35:387-397; Doig et al., 1997, Protein Science 6:147-155).

While structure (I) contains 22 specified amino acid residue positions,it is to be understood that the core peptides of the invention cancontain fewer than 22 amino acid residues. Indeed, truncated orinternally deleted forms of structure (I) containing as few as 18 oreven 15 amino acid residues that substantially retain the overallcharacteristics and properties of the amphipathic helix formed by thecore peptides of structure (I) are considered to be within the scope ofthe present invention.

Truncated forms of the peptides of structure (I) are obtained bydeleting one or more amino acids from the N-and/or C-terminus ofstructure (I). Internally deleted forms of structure (I) are obtained bydeleting one or more amino acids from internal positions within thepeptide of structure (I). The internal amino acid residues deleted mayor may not be consecutive residues.

Those of skill in the art will recognize that deleting an internal aminoacid residue from a core peptide of structure (I) will cause the planeof the hydrophilic-hydrophobic interface of the helix to rotate by 100°at the point of the deletion. As such rotations can significantly alterthe amphipathic properties of the resultant helix, in a preferredembodiment of the invention amino acid residues are deleted so as tosubstantially retain the alignment of the plane of thehydrophilic-hydrophobic interface along the entire long axis of thehelix.

This can be conveniently achieved by deleting a sufficient number ofconsecutive or non-consecutive amino acid residues such that onecomplete helical turn is deleted. An idealized α-helix contains 3.6residues per turn. Thus, in a preferred embodiment, groups of 3-4consecutive or non-consecutive amino acid residues are deleted. Whether3 amino acids or 4 amino acids are deleted will depend upon the positionwithin the helix of the first residue to be deleted. Determining theappropriate number of consecutive or non-consecutive amino acid residuesthat constitute one complete helical turn from any particular startingpoint within an amphipathic helix is well within the capabilities ofthose of skill in the art.

Due to the surmised importance of the basic cluster at the C-terminus ofthe core peptides of structure (I) in stabilizing the helix and theimportance of the hydrophobic cluster in effecting lipid binding andLCAT activation, in preferred embodiments of the invention, residuescomprising the basic and hydrophobic clusters are not deleted. Thus, inpreferred embodiments, residues 18, 19, 20 and 22 (basic cluster) andresidues 3, 6, 9 and 10 (hydrophobic cluster) are not deleted.

The core peptides of structure (I) can also be extended at one or bothtermini or internally with additional amino acid residues that do notsubstantially interfere with, and in some embodiments even enhance, thestructural and/or functional properties of the peptides. Indeed,extended core peptides containing as many as 23, 25, 26, 29 or even moreamino acid residues are considered to be within the scope of the presentinvention. Preferably, such extended peptides will substantially retainthe net amphipathicity and other properties of the peptides of structure(I). Of course, it will be recognized that adding amino acids internallywill rotate the plane of the hydrophobic-hydrophilic interface at thepoint of the insertion in a manner similar to that described above forinternal deletions. Thus, the considerations discussed above inconnection with internal deletions apply to internal additions, as well.

In one embodiment, the core peptides are extended at the N- and/orC-terminus by least one helical turn. Preferably, such extensions willstabilize the helical secondary structure in the presence of lipids,such as the end-cap amino acids and segments previously described.

In a particularly preferred embodiment, the core peptide of structure(I) is extended at the C-terminus by a single basic amino acid residue,preferably Lys (K). When so extended, X₁ is preferably D-Pro (p) or Gly(G); X₂ is preferably Val (V); X₃ is preferably Leu (L); X₄ ispreferably Asp (D); X₅ is preferably Leu (L); X₆ is preferably Phe (F);X₇ is preferably Arg (R); X₈ is preferably Glu (E); X₉ is preferably Leu(L); X₁₀ is preferably Leu (L); X₁₁, is preferably Asn (N); X₁₂ ispreferably Glu (E); X₁₃ is preferably Leu (L); X₁₄ is preferably Leu(L); X₁₅ is preferably Glu (E); X₁₆ is preferably Ala (A); X₁₇ ispreferably Leu (L); X₁₈ is preferably Lys (K); X₁₉ is preferably Gln(Q); X₂₀ is preferably Lys (K); X₂₁ is preferably Leu (L); and/or X₂₂ ispreferably Lys (K).

Also included within the scope of the present invention are "blocked"forms of the ApoA-I agonist, i.e., forms of the ApoA-I agonists in whichthe N- and/or C-terminus is blocked with a moiety capable of reactingwith the N-terminal --NH₂ or C-terminal --C(O)OH. It has been discoveredthat removing the N- and/or C-terminal charges of the ApoA-I agonists ofthe invention containing 18 or fewer amino acid residues (bysynthesizing N-acylated peptide amides/ester/hydrazides/alcohols andsubstitutions thereof) results in agonists which approach, and in someembodiments even exceed, the activity of the unblocked form of theagonist. In some embodiments containing 22 or more amino acids, blockingthe N- or C-terminus results in ApoA-I agonists which exhibit loweractivity than the unblocked forms. However, blocking both the N- andC-termini of ApoA-I agonists composed of 22 or more amino acids isexpected to restore activity. Thus, in a preferred embodiment of theinvention, either the N- and/or C-terminus (preferably both termini) ofcore peptides containing 18 or fewer amino acids are blocked, whereasthe N- and C-termini of peptides containing 22 or more amino acids areeither both blocked or both unblocked. Typical N-terminal blockinggroups include RC(O)--, where R is --H, (C₁ -C₆) alkyl, (C₁ -C₆)alkenyl, (C₁ -C₆) alkynyl, (C₅ -C₂₀) aryl, (C₆ -C₂₆) alkaryl, 5-20membered heteroaryl or 6-26 membered alkheteroaryl. Preferred N-terminalblocking groups include acetyl, formyl and dansyl. Typical C-terminalblocking groups include --C(O)NRR and --C(O)OR, where each R isindependently defined as above. Preferred C-terminal blocking groupsinclude those where each R is independently methyl. While not intendingto be bound by any particular theory, it is believed that such terminalblocking groups stabilize the α-helix in the presence of lipids (see,e.g., Venkatachelapathi et al., 1993, PROTEINS: Structure, Function andGenetics 15:349-359).

The native structure of ApoA-I contains eight helical units that arethought to act in concert to bind lipids (Nakagawa et al., 1985, J. Am.Chem. Soc. 107:7087-7092; Anantharamaiah et al., 1985, J. Biol. Chem.260:10248-10262; Vanloo et al., 1991, J. Lipid Res. 32:1253-1264; Mendezet al., 1994, J. Clin. Invest. 94:1698-1705; Palgunari et al., 1996,Arterioscler. Thromb. Vasc. Biol. 16:328-338; Demoor et al., 1996, Eur.J. Biochem. 239:74-84). Thus, also included in the present invention areApoA-I agonists comprised of dimers, trimers, tetramers and even higherorder polymers ("multimers") of the core peptides described herein. Suchmultimers may be in the form of tandem repeats, branched networks orcombinations thereof. The core peptides may be directly attached to oneanother or separated by one or more linkers.

The core peptides that comprise the multimers may be the peptides ofstructure (I), analogues of structure (I), mutated forms of structure(I), truncated or internally deleted forms of structure (I), extendedforms of structure (I) and/or combinations thereof. The core peptidescan be connected in a head-to-tail fashion (i.e., N-terminus toC-terminus), a head-to-head fashion, (i.e., N-terminus to N-terminus), atail-to-tail fashion (i.e., C-terminus to C-terminus), or combinationsthereof.

In one embodiment of the invention, the multimers are tandem repeats oftwo, three, four and up to about ten core peptides. Preferably, themultimers are tandem repeats of from 2 to 8 core peptides. Thus, in oneembodiment, the ApoA-I agonists of the invention comprise multimershaving the following structural formula:

    HH.brket open-st.LL.sub.m --HH.brket close-st..sub.n LL.sub.m --HH (II)

wherein:

each m is independently an integer from 0 to 1, preferably 1;

n is an integer from 0 to 10, preferably 0 to 8;

each "HH" independently represents a core peptide or peptide analogue ofstructure (I) or a mutated, truncated, internally deleted or extendedform thereof as described herein;

each "LL" independently represents a linker; and

each "-" independently designates a covalent linkage.

In structure (II), the linker LL can be any bifunctional moleculecapable of covalently linking two peptides to one another. Thus,suitable linkers are bifunctional molecules in which the functionalgroups are capable of being covalently attached to the N- and/orC-terminus of a peptide. Functional groups suitable for attachment tothe N- or C-terminus of peptides are well known in the art, as aresuitable chemistries for effecting such covalent bond formation.

The linker may be flexible, rigid or semi-rigid, depending on thedesired properties of the multimer. Suitable linkers include, forexample, amino acid residues such as Pro or Gly or peptide segmentscontaining from about 2 to about 5, 10, 15 or 20 or even more aminoacids, bifunctional organic compounds such as H₂ N(CH₂)_(n) COOH where nis an integer from 1 to 12, and the like. Examples of such linkers, aswell as methods of making such linkers and peptides incorporating suchlinkers are well-known in the art (see, e.g., Hunig et al., 1974, Chem.Ber. 100:3039-3044; Basak et al., 1994, Bioconjug. Chem. 5(4):301-305).

In a preferred embodiment of the invention, the tandem repeats areinternally punctuated by a single proline residue. To this end, in thoseinstances where the core peptides are terminated at their N- orC-terminus with proline, such as, e.g., where X₁ in structure (I) is Pro(P) or D-Pro (p), m in structure (II) is preferably 0. In thoseinstances where the core peptides do not contain an N- or C-terminalproline, LL is preferably Pro (P) or D-Pro (p) and m is preferably 1.

In certain embodiments of the invention, it may be desirable to employcleavable linkers that permit the release of one or more helicalsegments (HH) under certain conditions. Suitable cleavable linkersinclude peptides having amino acid sequences that are recognized byproteases, oligonucleotides that are cleaved by endonucleases andorganic compounds that can be cleaved via chemical means, such as underacidic, basic or other conditions. Preferably, the cleavage conditionswill be relatively mild so as not to denature or otherwise degrade thehelical segments and/or non-cleaved linkers composing the multimericApoA-I agonists.

Peptide and oligonucleotide linkers that can be selectively cleaved, aswell as means for cleaving the linkers are well known and will bereadily apparent to those of skill in the art. Suitable organic compoundlinkers that can be selectively cleaved will be apparent to those ofskill in the art, and include those described, for example, in WO94/08051, as well as the references cited therein.

In a preferred embodiment, the linkers employed are peptides that aresubstrates for endogenous circulatory enzymes, thereby permitting themultimeric ApoA-I agonists to be selectively cleaved in vivo. Endogenousenzymes suitable for cleaving the linkers include, for example,proapolipoprotein A-I propeptidase. Appropriate enzymes, as well aspeptide segments that act as substrates for such enzymes, are well-knownin the art (see, e.g., Edelstein et al., 1983, J. Biol. Chem.258:11430-11433; Zanis, 1983, Proc. Natl. Acad. Sci. USA 80:2574-2578).

As discussed above, a key feature of the core peptides of the inventionis their ability to form intermolecular hydrogen-bonds or salt bridgeswhen arranged in an antiparallel fashion. Thus, in a preferredembodiment of the invention, linkers of sufficient length andflexibility are used so as to permit the helical segments (HH) ofstructure (II) to align in an antiparallel fashion and formintermolecular hydrogen-bonds or salt bridges in the presence of lipids.

Linkers of sufficient length and flexibility include, but are notlimited to, Pro (P), Gly (G), Cys-Cys, H₂ N-(CH₂)_(n) --COOH where n is1 to 12, preferably 4 to 6; H₂ N-aryl-COOH and carbohydrates.

Alternatively, as the native apolipoproteins permit cooperative bindingbetween antiparallel helical segments, peptide linkers which correspondin primary sequence to the peptide segments connecting adjacent helicesof the native apolipoproteins, including, for example, ApoA-I, ApoA-II,ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE and ApoJ can beconveniently used to link the core peptides. These sequences are wellknown in the art (see, e.g., Rosseneu et al., "Analysis of the Primaryand of the Secondary Structure of the Apolipoproteins," In: Structureand Function of Lipoproteins, Ch. 6, 159-183, CRC Press, Inc., 1992).

Other linkers which permit the formation of intermolecular hydrogenbonds or salt bridges between tandem repeats of antiparallel helicalsegments include peptide reverse turns such as β-turns and γ-turns, aswell as organic molecules that mimic the structures of peptide β-turnsand/or γ-turns. Generally, reverse turns are segments of peptide thatreverse the direction of the polypeptide chain so as to allow a singlepolypeptide chain to adopt regions of antiparallel β-sheet orantiparallel α-helical structure. β-turns generally are composed of fouramino acid residues and γ-turns are generally composed of three aminoacid residues.

The conformations and sequences of many peptide β-turns have beenwell-described in the art and include, by way of example and notlimitation, type-I, type-I', type-II, type-II', type-III, type-III',type-IV, type-V, type-V', type-VIa, type-VIb, type-VII and type-VIII(see, Richardson, 1981, Adv. Protein Chem. 34:167-339; Rose et al.,1985, Adv. Protein Chem. 37:1-109; Wilmot et al., 1988, J. Mol. Biol.203:221-232; Sibanda et al., 1989, J. Mol. Biol. 206:759-777; Tramontanoet al., 1989, Proteins: Struct. Funct. Genet. 6:382-394).

The specific conformations of short peptide turns such as β-turns dependprimarily on the positions of certain amino acid residues in the turn(usually Gly, Asn or Pro). Generally, the type-I β-turn is compatiblewith any amino acid residue at positions 1 through 4 of the turn, exceptthat Pro cannot occur at position 3. Gly predominates at position 4 andPro predominates at position 2 of both type-I and type-II turns. Asp,Asn, Ser and Cys residues frequently occur at position 1, where theirside chains often hydrogen-bond to the NH of residue 3.

In type-II turns, Gly and Asn occur most frequently at position 3, asthey adopt the required backbone angles most easily. Ideally, type-I'turns have Gly at positions 2 and 3, and type-II' turns have Gly atposition 2. Type-III turns generally can have most amino acid residues,but type-III' turns usually require Gly at positions 2 and 3. Type-VIaand VIb turns generally have a cis peptide bond and Pro as an internalresidue. For a review of the different types and sequences of β-turns inproteins and peptides the reader is referred to Wilmot et al., 1988, J.Mol. Biol. 203:221-232.

The conformation and sequences of many peptide γ-turns have also beenwell-described in the art (see, e.g., Rose et al., 1985, Adv. ProteinChem. 37:1-109; Wilmer-White et al., 1987, Trends Biochem. Sci.12:189-192; Wilmot et al., 1988, J. Mol. Biol. 203:221-232; Sibanda etal., 1989, J. Mol. Biol. 206:759-777; Tramontano et al., 1989, Proteins:Struct. Funct. Genet. 6:382-394). All of these types of β-turns andγ-turn structures and their corresponding sequences, as well as laterdiscovered peptide β-turns and γ-turn structures and sequences, arespecifically contemplated by the invention.

Alternatively, the linker (LL) may comprise an organic molecule ormoiety that mimics the structure of a peptide β-turn or γ-turn. Suchβ-turn and/or γ-turn mimetic moieties, as well as methods forsynthesizing peptides containing such moieties, are well known in theart, and include, among others, those described in Giannis and Kolter,1993 Angew. Chem. Intl. Ed. Eng. 32:1244-1267; Kahn et al., 1988, J.Molecular Recognition 1:75-79; and Kahn et al., 1987, Tetrahedron Lett.28:1623-1626.

In still another embodiment of the invention, the multimers are in theform of branched networks (see, e.g., FIG. 7). Such networks areconveniently obtained through the use of multifunction linking moietiesthat permit more than two helical units to be attached to a simplelinking moiety. Thus, branched networks employ molecules having three,four or even more functional groups that are capable of covalentlyattaching to the N- and/or C-terminus of a peptide. Suitable linkingmoieties include, for example, amino acid residues having side chainsbearing hydroxyl, sulfanyl, amino, carboxyl, amide and/or esterfunctionalities, such as, for example, Ser (S), Thr (T), Cys (C), Tyr(Y), Asn (N), Gln (Q), Lys (K), Arg (R), Orn, Asp (D) and Glu (E); orother organic molecules containing such functional groups.

The helical segments attached to a single linking moiety need not beattached via like termini. Indeed, in some embodiments the helicalsegments are attached to a single linking moiety so as to be arranged inan antiparallel fashion, i.e., some of the helices are attached viatheir N-termini, others via their C-termini.

The helical segments can be attached directly to the linking moiety, ormay be spaced from the linking moiety by way of one or more bifunctionallinkers (LL), as previously described.

Referring to FIGS. 7A and 7B, it can be seen that a branched network canbe described in terms of the number of "nodes" comprising the network,where each multifunctional linking moiety constitutes a node. In FIGS.7A and 7B, helical segments (i.e., core peptides of the invention) areillustrated as cylinders, and multifunctional linking moieties (ornodes) as circles (), where the number of lines emanating from thecircle indicates the "order" (or number of functional groups) of themultifunctional linking moiety.

The number of nodes in the network will generally depend on the totaldesired number of helical segments, and will typically be from about 1to 2. Of course, it will be appreciated that for a given number ofdesired helical segments, networks having higher order linking moietieswill have fewer nodes. For example, referring to FIGS. 7A and 7B, atertiary-order network (i.e., a network having trifunctional linkingmoieties) of seven helical units has three nodes (FIG. 7A), whereas aquaternary order network (i.e., a network having tetrafunctional linkingmoieties) of seven helical units has only two nodes (FIG. 7B).

The networks may be of uniform order, i.e., networks in which all nodesare, for example, trifunctional or tetrafunctional linking moieties, ormay be of mixed order, e.g., networks in which the nodes are mixturesof, for example, trifunctional and tetrafunctional linking moieties. Ofcourse, it is to be understood that even in uniform order networks thelinking moieties need not be identical. A tertiary order network mayemploy, for example, two, three, four or even more differenttrifunctional linking moieties.

Like the linear multimers, the helical segments comprising the branchednetwork may be, but need not be, identical.

An example of such a mixed order branched network is illustrated in FIG.7C. In FIG. 7C, helical segments (i.e., core peptides of the invention)are illustrated as cylinders and multifunctional linking moieties ascircles (), where the number of lines emanating from the circleindicates the "order" (or number of functional groups) of themultifunctional linking moiety. Lines connecting helical segmentsrepresent bifunctional linkers LL, as previously described. Helicalsegments which comprise the branched networks may be tandem repeats ofcore peptides, as previously described.

In one illustrative embodiment, the branched networks of the inventionare described by the formula:

    X--N.sub.ya --X.sub.(ya-1) .paren open-st.N.sub.yb --X.sub.(yb-1)).sub.p (III)

wherein:

each X is independently HH.paren open-st.LL_(m) --HH.paren close-st._(n)LL_(m) --HH;

each HH is independently a core peptide of structure (I) or an analogueor mutated, truncated, internally deleted or extended form thereof asdescribed herein;

each LL is independently a bifunctional linker;

each m is independently an integer from 0 to 1;

each n is independently an integer from 0 to 8;

N_(ya) and N_(yb) are each independently a multifunctional linkingmoiety where y_(a) and y_(b) represent the number of functional groupson N_(ya) and N_(yb), respectively;

each y_(a) or y_(b) is independently an integer from 3 to 8;

p is an integer from 0 to 7; and

each "-" independently designates a covalent bond.

In a preferred embodiment, the branched network comprises a "Lys-tree,"i.e., a network wherein the multifunctional linking moiety is one ormore Lys (K) residues (see, e.g., FIG. 7D).

In one illustrative embodiment, the "Lys tree" branched networks of theinvention are described by the formulae: ##STR1## wherein:

each X is independently HH.paren open-st.LL_(m) --HH.paren close-st._(n)LL_(m) --HH;

each HH is independently a core peptide or peptide analogue of structure(I) or a mutated, truncated, internally deleted or extended form thereofas described herein;

each LL is independently a bifunctional linker;

each n is independently an integer from 0 to 8;

each m is independently an integer from 0 to 1;

R₁ is --OR or --NRR; and

each R is independently --H, (C₁ -C₆) alkyl, (C₁ -C₆) alkenyl, (C₁ -C₆)alkynyl; (C₅ -C₂₀) aryl (C₆ -C₂₆) alkaryl, 5-20 membered heteroaryl or6-26 membered alkheteroaryl.

5.1.1. ANALYSIS OF STRUCTURE AND FUNCTION

The structure and function of the core peptides or peptide analogues ofthe invention, as well as ApoA-I agonists composed of such corepeptides, including the multimeric forms described above, can be assayedin order to select active agonists or mimetics of ApoA-I. For example,the core peptides or peptide analogues can be assayed for their abilityto form α-helices in the presence of lipids, to bind lipids, to formcomplexes with lipids, to activate LCAT, to promote cholesterol efflux,etc.

Methods and assays for analyzing the structure and/or function of thepeptides are well-known in the art. Preferred methods are provided inthe working examples, infra. For example, the circular dichroism (CD)and nuclear magnetic resonance (NMR) assays described in Section 7,infra, can be used to analyze the structure of the peptides or peptideanalogues--particularly the degree of helicity in the presence oflipids. The ability to bind lipids can be determined using thefluorescence spectroscopy assay described in Section 7, infra. Theability of the peptides and/or peptide analogues to activate LCAT can bereadily determined using the LCAT activation described in Section 8,infra. The in vitro and in vivo assays described in Section 9, 10 and11, infra, can be used to evaluate the half-life, distribution,cholesterol efflux and effects on RCT.

Generally, core peptides and/or peptide analogues according to theinvention which exhibit the properties listed in TABLE IV, infra, areconsidered to be active.

                  TABLE IV                                                        ______________________________________                                        PROPERTIES OF ACTIVE PEPTIDES                                                                               Preferred                                         Range Range                                                                 ______________________________________                                        % Helicity in the presence of                                                                     ≧60%                                                                             ≧80%                                       lipids (Ri = 30)                                                              (unblocked 22-amino acid residue                                              peptides)                                                                     % Helicity in the presence of ≧40% ≧60%                         lipids (Ri = 30)                                                              (unblocked 18-amino acid residue                                              peptides)                                                                     % Helicity in the presence of ≧60% ≧80%                         lipids (Ri = 30)                                                              (blocked 18-amino acid residue                                                peptides and shorter peptides)                                                Lipid Binding (in the presence of 0.5-10 μM                                SUVs) peptide                                                                  R.sub.i = 1-50                                                               LCAT activation ≧38% ≧80%                                     ______________________________________                                         R.sub.i is lipid:peptide molar ratio                                     

As illustrated in the working examples, infra, core peptides whichexhibit a high degree of LCAT activation (≧38%) generally possesssignificant α-helical structure in the presence of lipidic smallunilamellar vesicles (SUVs) (≧60% helical structure in the case ofunblocked peptides containing 22 or more amino acid residues and blockedpeptides containing 18 or fewer amino acid residues; ≧40% helicalstructure in the case of unblocked peptides containing 18 or fewer aminoacids), and those peptides which exhibit little or no LCAT activationpossess little α-helical structure. However, in certain instances,peptides which exhibit significant helical structure in the presence oflipids do not effect significant LCAT.

Similarly, while core peptides that exhibit significant LCAT activationtypically bind lipids, in certain instances peptides which exhibit lipidbinding do not effect significant LCAT activation.

As a consequence, it will be recognized by those of skill in the artthat while the ability of the core peptides described herein to formα-helices (in the presence of lipids) and to bind lipids is critical foractivity, in many instances these properties may not be sufficient.Thus, in a preferred embodiment core peptides of the invention aresubjected to a series of screens to select for core peptides exhibitingsignificant pharmacological activity.

In a first step, a core peptide is screened for its ability to form anα-helix in the presence of lipids using the CD assay described inSection 7, infra. Those peptides which are at least 40% helical(unblocked peptides containing 18 or fewer amino acids) or 60% helical(blocked peptides containing 18 or fewer amino acids; unblocked peptidescontaining 22 or more amino acids) in the presence of lipids (at a conc.of about 5 μM and a lipid:peptide molar ratio of about 30) are thenscreened for their ability to bind lipids using the fluorescence assaydescribed in Section 7, infra. Of course, only those core peptides whichcontain a fluorescent Trp (W) or Nal residue are screened for lipidbinding via fluorescence. However, for peptides which do not containfluorescent residues, binding to lipids is obvious when helicityincreases in the presence of lipids.

Core peptides which exhibit lipid binding in the presence of SUVs(0.5-10 μM peptide; lipid:peptide molar ratio in the range of 1 to 50)are then screened for pharmacological activity. Of course, thepharmacological activity screened for will depend upon the desired useof the ApoA-I agonists. In a preferred embodiment, the core peptides arescreened for their ability to activate LCAT, as peptides which activateLCAT are particularly useful in the methods described herein. Corepeptides which exhibit at least about 38% LCAT activation as comparedwith native human ApoA-I (as determined using the LCAT activation assaydescribed in Section 8, infra), are preferred, with core peptidesexhibiting 50%, 60%, 70%, 80% or even 90% or more being particularlypreferred.

5.1.2. PREFERRED EMBODIMENTS

The ApoA-I agonists of the invention can be further defined by way ofpreferred embodiments.

In one preferred embodiment, the ApoA-I agonists are 22 amino acidresidue peptides according to structure (I), or the N-terminal acylatedand/or C-terminal amidated or esterified forms thereof.

In another preferred embodiment, the ApoA-I agonists are 22 amino acidresidue peptides according to structure (I), or the N-terminal acylatedand/or C-terminal amidated or esterified forms thereof, in which X₇ is abasic amino acid, Asn (N) or Glu (E); X₈ is an acidic amino acid or Arg(R); X₁₂ is an acidic amino acid or Asn (N); and/or X₁₅ is an acidicamino acid, Gln (Q) or Lys (K); and X₁, X₂, X₃, X₄, X₅, X₆, X₉, X₁₀,X₁₁, X₁₃, X₁₄, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀ and X₂₁ are as previously definedfor Structure (I).

In another preferred embodiment, the ApoA-I agonists are 22 amino acidresidue peptides according to structure (I), or the N-terminal acylatedand/or C-terminal amidated or esterified forms thereof, in which:

X₁ is Pro (P), Gly (G), Ala (A), Gln (Q), Asn (N), Asp (D) or D-Pro (p);

X₂ is Ala (A), Val (V) or Leu (L);

X₄ is Asp (D) or Glu (E);

X₇ is Lys (K), Arg (R), Orn, Asn (N) or Glu (E);

X₈ is Asp (D), Arg (R) or Glu (E);

X₁₁ is Asn (N), Gln (Q), Glu (E) or Arg (R);

X₁₂ is Asp (D), Glu (E) or Asn (N);

X₁₃ is Leu (L), Gly (G) or Aib;

X₁₅ is Asp (D), Glu (E), Gln (Q) or Lys (K);

X₁₆ is Ala (A), Trp (W), Gly (G), Leu (L), Phe (F) or Nal;

X₁₇ is Leu (L), Gly (G) or Nal;

X₁₈ is Lys (K), Orn, Gln (Q) or Asn (N);

X₁₉ is Lys (K), Orn, Gln (Q) or Asn (N);

X₂₀ is Lys (K) or Orn;

X₂₁ is Leu (L); and/or

X₂₂ is Lys (K) or Orn, and X₃, X₅, X₆, X₉, X₁₀ and X₁₄ are as previouslydefined for structure (I).

An even more preferred embodiment according to this aspect of theinvention are those peptides in which:

X₂ is Val (V);

X₃ is Leu (L);

X₅ is Leu (L);

X₆ is Phe (F);

X₇ is Arg (R) or Lys (K);

X₈ is Glu (E);

X₉ is Leu (L);

X₁₀ is Leu (L);

X₁₁ is Asn (N) or Glu (Q);

X₁₂ is Glu (E); and/or

X₁₅ is Glu (E);

and X₁, X₄, X₁₃, X₁₄, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁ and X₂₂ are aspreviously defined for structure (I) or are as defined in the precedingparagraph.

In yet another preferred embodiment, the ApoA-I agonists are 22 aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich only one of X₁₈ or X₁₉ is a basic amino acid and the other one ofX₁₈ or X₁₉ is Gln (Q) or Asn (N).

In yet another preferred embodiment, the ApoA-I agonists are peptidesaccording to structure (I), or the N-terminal acylated and/or C-terminalamidated or esterified forms thereof, in which one of X₁₈ or X₁₉ is Lys(K) or Orn and the other one of X₁₈ or X₁₉ is Gln (Q) or Asn (N).

In still another preferred embodiment, the ApoA-I agonists are 22 aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich one of X₉, X₁₀, X₁₃, X₁₄, X₁₆ or X₁₇ is Gly (G) and the others areother than Gly (G).

In still another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich X₁₃ is Gly (G) and each of X₉, X₁₀, X₁₄, X₁₆ and X₁₇ is other thanGly (G).

In yet another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich:

X₁ is Pro (P), Gly (G) or D-Pro (p);

X₂ is Val (V);

X₃ is Leu (L);

X₄ is Asp (D) or Glu (E);

X₅ is L (Leu) or Phe (F);

X₆ is Phe (F);

X₇ is Arg (R);

X₈ is Glu (E);

X₉ is Leu (L);

X₁₀ is Leu (L) or Trp (W);

X₁₁ is Asn (N);

X₁₂ is Glu (E);

X₁₃ is Gly (G);

X₁₄ is Leu (L);

X₁₅ is Glu (E);

X₁₆ is Ala (A) or Trp (W);

X₁₇ is Leu (L) or Nal;

X₁₈ is Lys (K) or Orn;

X₁₉ is Gln (Q);

X₂₀ is Lys (K) or Orn;

X₂₁ is Leu (L); and

X₂₂ is Lys (K) or Orn.

Particularly preferred ApoA-I agonists according to this aspect of theinvention are selected from the group consisting of:

    ______________________________________                                        peptide 3                                                                              PVLDLFRELLNEGLEALKQKLK                                                                          (SEQ ID NO:3);                                     peptide 13        GVLDLFRELLNEGLEALKQKLK                (SEQ ID NO:13);         - peptide 19        pVLDLFRELLNEGLEALKQKLK         (SEQ ID NO:19);            - peptide 137       PVLDLFRELLNEGLEAZKQKLK         (SEQ ID NO:137);           - peptide 138       PVLDLFRELLNEGLEWLKQKLK         (SEQ ID NO:138);           - peptide 139       PVLDLFRELWNEGLEALKQKLK         (SEQ ID NO:139);           - peptide 140       PVLDLFRELLNEGLEALOQOLO         (SEQ ID NO:140);           - peptide 141       PVLDFFRELLNEGLEALKQKLK         (SEQ ID NO:141);           - peptide 142       PVLELFRELLNEGLEALKQKLK         (SEQ ID NO:142);         ______________________________________                                    

and the C-terminal amidated or esterified and/or N-terminal acylatedforms thereof.

In still another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich X₉ is Gly (G) and each of X₁₀, X₁₃, X₁₄, X₁₆ and X₁₇ is other thanGly (G). A particularly preferred ApoA-I agonist according to thisaspect of the invention is peptide 20:

    PVLDLFREGLNELLEALKQKLK (SEQ ID NO:20).

In still another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich X₁₀ is Gly (G) and each of X₉, X₁₃, X₁₄, X₁₆ and X₁₇ is other thanGly (G). A particularly preferred ApoA-I agonist according to thisaspect of the invention is peptide 9: PVLDLFRELGNELLEALKQKLK (SEQ IDNO:9).

In still another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich X₁₄ is Gly (G) and each of X₉, X₁₀, X₁₃, X₁₆ and X₁₇ is other thanGly (G). A particularly preferred ApoA-I agonist according to thisaspect of the invention is peptide 126:

    PVLDLFRELLNELGEALKQKLK (SEQ ID NO:126).

In still another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich X₁₆ is Gly (G) and each of X₉, X₁₀, X₁₃, X₁₄ and X₁₇ is other thanGly (G). A particularly preferred ApoA-I agonist according to thisaspect of the invention is peptide 22:

    PVLDLFRELLNELLEGLKQKLK (SEQ ID NO:22).

In still another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich X₁₇ is Gly (G) and each of X₉, X₁₀, X₁₃, X₁₄ and X₁₆ is other thanGly (G). A particularly preferred ApoA-I agonist according to thisaspect of the invention is peptide 12:

    PVLDLFRELLNELLEAGKQKLK (SEQ ID NO:12).

Embodiments containing internal glycine residues can be readilysynthesized in high yield by way of segment condensation, therebyproviding significant advantages for large-scale production. Segmentcondensation, i.e., the joining together of small constituent peptidechains to form a larger peptide chain, has been used to prepare manybiologically active peptides, including 44-amino acid residue mimics ofApoA-I (see, e.g., Nakagawa et al., 1985, J. Am Chem. Soc.107:7087-7083; Nokihara et al., 1989, Peptides 1988:166-168;Kneib-Cordonnier et al., 1990, Int. J. Pept. Protein Res. 35:527-538),and is considered to be the most cost-effective method for high-yieldbulk synthesis of the core peptides of the invention.

Advantages of synthesis via segment condensation include the ability tocondense pre-formed segments in the solution phase and the ease ofpurification of the final product. Drawbacks of the method include lowcoupling efficiency and yield at the condensation step and lowsolubility of certain peptide sequences.

The coupling efficiency of the condensation step can be significantlyincreased by increasing the coupling time. Typically, increasing thecoupling time results in increased racemezation of the product (Sieberet al., 1970, Helv. Chim. Acta 53:2135-2150). However, since glycinelacks a chiral center it does not undergo racemezation (prolineresidues, due to steric hindrance, also undergo little or noracemezation at long coupling times). Thus, embodiments containinginternal glycine residues can be synthesized in bulk in high yield viasegment condensation by synthesizing constituent segments which takeadvantage of the fact that glycine residues do not undergo racemezation.Thus, embodiments containing internal glycine residues providesignificant synthetic advantages for large-scale bulk preparation.

In yet another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich each of X₉, X₁₀, X₁₃, X₁₄, X₁₆ and X₁₇ is other than Gly (G).

In yet another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich:

X₁ is Pro (P), Gly (G), Ala (A) or D-Pro (p);

X₂ is Val (V) or Leu (L);

X₃ is Leu (L);

X₄ is Asp (D) or Glu (E);

X₅ is Leu (L) or Phe (F);

X₆ is Leu (L) or Phe (F);

X₇ is Arg (R) or Lys (K);

X₈ is Glu (E);

X₉ is Leu (L);

X₁₀ is Leu (L) or Trp (W);

X₁₁ is Asn (N) or Gln (Q);

X₁₂ is Glu (E);

X₁₃ is Leu (L) or Aib;

X₁₄ is Leu (L), Trp (W) or Nal;

X₁₅ is Glu (E);

X₁₆ is Ala (A), Leu (L), Trp (W) or Nal;

X₁₇ is Leu (L) or Nal;

one of X₁₈ or X₁₉ is Gln (Q) and the other is Lys (K) or Orn;

X₂₀ is Lys (K) or Orn;

X₂₁ is Leu (L); and

X₂₂ is Lys (K) or Orn.

In a particularly preferred embodiment according to this aspect of theinvention, X₂ is Val (V); X₄ is Asp (D); X₅ is Leu (L); X₆ is Phe (F);X₇ is Arg R); X₁₀ is Leu (L); X₁₁ is Asn (N); X₁₃ is Leu (L); X₁₄ is Leu(L); X₁₆ is Ala (A); X₁₇ is Leu (L); X₁₈ is Lys (K); X₁₉ is Gln (Q); X₂₀is Lys (K) and/or X₂₂ is Lys (K).

In still another preferred embodiment, the ApoA-I agonists are alteredor mutated forms of the peptides of structure (I), or the N-terminalacylated and/or C-terminal amidated or esterified forms thereof, inwhich:

X₁ is other than Aib, Val (V) or Leu (L);

X₂ is other than D-Val (v);

X₅ is other than Lys (K), Glu (E), Trp (W) or Nal;

X₆ is other than Trp (W);

X₇ is other than Trp (W) or Leu (L);

X₈ is other than Trp (W);

X₉ is other than Lys (K) or Trp (W);

X₁₁ is other than Trp (W);

X₁₂ is other than Trp (W) or Leu (L);

X₁₃ is other than Glu (E) or Trp (W);

X₁₅ is other than Trp (W); and/or

X₂₁ is other than Lys (K).

In yet another preferred embodiment, the ApoA-I agonists of theinvention are selected from the group of peptides set forth below:

    ______________________________________                                        peptide 1                                                                              PVLDLFRELLNELLEZLKQKLK                                                                          (SEQ ID NO:1);                                     peptide 2          GVLDLFRELLNELLEALKQKLKK (SEQ ID NO:2);                        - peptide 3    PVLDLFRELLNELLEWLKQKLK  (SEQ ID NO:3);                         - peptide 4    PVLDLFRELLNELLEALKQKLK  (SEQ ID NO:4);                         - peptide 5    pVLDLFRELLNELLEALKQKLKK (SEQ ID NO:5);                         - peptide 6    PVLDLFRELLNEXLEALKQKLK  (SEQ ID NO:6);                         - peptide 7    PVLDLFKELLNELLEALKQKLK  (SEQ ID NO:7);                         - peptide 8    PVLDLFRELLNEGLEALKQKLK  (SEQ ID NO:8);                         - peptide 9    PVLDLFRELGNELLEALKQKLK  (SEQ ID NO:9);                         - peptide 10   PVLDLFRELLNELLEAZKQKLK  (SEQ ID NO:10);                        - peptide 11   PVLDLFKELLQELLEALKQKLK  (SEQ ID NO:11);                        - peptide 12   PVLDLFRELLNELLEAGKQKLK  (SEQ ID NO:12);                        - peptide 13   GVLDLFRELLNEGLEALKQKLK  (SEQ ID NO:13);                        - peptide 14   PVLDLFRELLNELLEALOQOLO  (SEQ ID NO:14);                        - peptide 15   PVLDLFRELWNELLEALKQKLK  (SEQ ID NO:15);                        - peptide 16   PVLDLLRELLNELLEALKQKLK  (SEQ ID NO:16);                        - peptide 17   PVLELFKELLQELLEALKQKLK  (SEQ ID NO:17);                        - peptide 18   GVLDLFRELLNELLEALKQKLK  (SEQ ID NO:18);                        - peptide 19   pVLDLFRELLNEGLEALKQKLK  (SEQ ID NO:19);                        - peptide 20   PVLDLFREGLNELLEALKQKLK  (SEQ ID NO:20);                        - peptide 21   pVLDLFRELLNELLEALKQKLK  (SEQ ID NO:21);                        - peptide 22   PVLDLFRELLNELLEGLKQKLK  (SEQ ID NO:22);                        - peptide 23   PLLELFKELLQELLEALKQKLK  (SEQ ID NO:23);                        - peptide 24   PVLDLFRELLNELLEALQKKLK  (SEQ ID NO:24);                        - peptide 25   PVLDFFRELLNEXLEALKQKLK  (SEQ ID NO:25);                        - peptide 26   PVLDLFRELLNELLELLKQKLK  (SEQ ID NO:26);                        - peptide 27   PVLDLFRELLNELZEALKQKLK  (SEQ ID NO:27);                        - peptide 28  PVLDLFRELLNELWEALKQKLK   (SEQ ID NO:28);                        - peptide 29  AVLDLFRELLNELLEALKQKLK   (SEQ ID NO:29);                        - peptide 123 QVLDLFRELLNELLEALKQKLK   (SEQ ID NO:123);                       - peptide 124 PVLDLFQELLNELLEALOQOLO   (SEQ ID NO:124);                       - peptide 125 NVLDLFRELLNELLEALKQKLK   (SEQ ID NO:125);                       - peptide 126 PVLDLFRELLNELGEALKQKLK   (SEQ ID NO:126);                       - peptide 127 PVLDLFRELLNELLELLKQKLK   (SEQ ID NO:127);                       - peptide 128 PVLDLFRELLNELLEFLKQKLK   (SEQ ID NO:128);                       - peptide 129 PVLELFNDLLRELLEALQKKLK   (SEQ ID NO:129);                       - peptide 130 PVLELFNDLLRELLEALKQKLK   (SEQ ID NO:130);                       - peptide 131 PVLELFKELLNELLDALRQKLK   (SEQ ID NO:131);                       - peptide 132 PVLDLFRELLENLLEALQKKLK   (SEQ ID NO:132);                       - peptide 133 PVLELFERLLEDLLQALNKKLK   (SEQ ID NO:133);                       - peptide 134 PVLELFERLLEDLLKALNQKLK   (SEQ ID NO:134);                       - peptide 135 DVLDLFRELLNELLEALKQKLK   (SEQ ID NO:135);                       - peptide 136 PALELFKDLLQELLEALKQKLK   (SEQ ID NO:136);                       - peptide 137 PVLDLFRELLNEGLEAZKQKLK   (SEQ ID NO:137);                       - peptide 138 PVLDLFRELLNEGLEWLKQKLK   (SEQ ID NO:138);                       - peptide 139 PVLDLFRELWNEGLEALKQKLK   (SEQ ID NO:139);                       - peptide 140 PVLDLFRELLNEGLEALOQOLO   (SEQ ID NO:140);                       - peptide 141 PVLDFFRELLNEGLEALKQKLK   (SEQ ID NO:141);                       - peptide 142 PVLELFRELLNEGLEALKQKLK   (SEQ ID NO:142);                    ______________________________________                                    

and the N-terminal acylated (particularly acetylated or dansylated)and/or C-terminal amidated or esterified forms thereof, wherein X isAib; Z is Nal; and O is Orn.

In still another preferred embodiment, the ApoA-I agonists of theinvention are selected from the group of peptides set forth below:

    ______________________________________                                        peptide 1                                                                              PVLDLFRELLNELLEZLKQKLK                                                                          (SEQ ID NO:1);                                     peptide 2  GVLDLFRELLNELLEALKQKLKK  (SEQ ID NO:2);                               - peptide 3  PVLDLFRELLNELLEWLKQKLK   (SEQ ID NO:3);                          - peptide 4  PVLDLFRELLNELLEALKQKLK   (SEQ ID NO:4);                          - peptide 5  pVLDLFRELLNELLEALKQKLKK  (SEQ ID NO:5);                          - peptide 6  PVLDLFRELLNEXLEALKQKLK   (SEQ ID NO:6);                          - peptide 7  PVLDLFKELLNELLEALKQKLK   (SEQ ID NO:7);                          - peptide 8  PVLDLFRELLNEGLEALKQKLK   (SEQ ID NO:8);                          - peptide 9  PVLDLFRELGNELLEALKQKLK   (SEQ ID NO:9);                          - peptide 10 PVLDLFRELLNELLEAZKQKLK   (SEQ ID NO:10);                         - peptide 11 PVLDLFKELLQELLEALKQKLK   (SEQ ID NO:11);                         - peptide 12 PVLDLFRELLNELLEAGKQKLK   (SEQ ID NO:12);                         - peptide 13 GVLDLFRELLNEGLEALKQKLK   (SEQ ID NO:13);                         - peptide 14 PVLDLFRELLNELLEALOQOLO   (SEQ ID NO:14);                         - peptide 15 PVLDLFRELWNELLEALKQKLK   (SEQ ID NO:15);                         - peptide 16 PVLDLLRELLNELLEALKQKLK   (SEQ ID NO:16);                         - peptide 17 PVLELFKELLQELLEALKQKLK   (SEQ ID NO:17);                         - peptide 18 GVLDLFRELLNELLEALKQKLK   (SEQ ID NO:18);                         - peptide 19 pVLDLFRELLNEGLEALKQKLK   (SEQ ID NO:19);                         - peptide 20 PVLDLFREGLNELLEALKQKLK   (SEQ ID NO:20);                         - peptide 21 pVLDLFRELLNELLEALKQKLK   (SEQ ID NO:21);                         - peptide 22 PVLDLFRELLNELLEGLKQKLK   (SEQ ID NO:22);                         - peptide 23 PLLELFKELLQELLEALKQKLK   (SEQ ID NO:23);                         - peptide 24 PVLDLFRELLNELLEALQKKLK   (SEQ ID NO:24);                         - peptide 25 PVLDFFRELLNEXLEALKQKLK   (SEQ ID NO:25);                         - peptide 26 PVLDLFRELLNELLELLKQKLK   (SEQ ID NO:26);                         - peptide 27 PVLDLFRELLNELZEALKQKLK   (SEQ ID NO:27);                         - peptide 28 PVLDLFRELLNELWEALKQKLK   (SEQ ID NO:28);                         - peptide 29 AVLDLFRELLNELLEALKQKLK   (SEQ ID NO:29);                      ______________________________________                                    

and the N-terminal acylated (particularly acetylated or dansylated)and/or C-terminal amidated or esterified forms thereof, wherein X isAib; Z is Nal; and O is Orn.

In yet another preferred embodiment, the ApoA-I agonists are multimericforms according to structures II, III and/or IV in which HH is a peptideaccording to structure (I), or an N-terminal acylated and/or C-terminalamidated or esterified form thereof, or any of the preferred peptidesaccording to structure (I) described herein.

In yet another preferred embodiment, the core peptides that compose theApoA-I agonists are not any of the following peptides:

    ______________________________________                                        peptide 75: PVLDEFREKLNEELEALKQKLK                                                                           (SEQ ID                                            NO:75);                                                                   peptide 94:   PVLDEFREKLNEALEALKQKLK   (SEQ ID                                    NO:94);                                                                      - peptide 109:  PVLDEFREKLNERLEALKQKLK   (SEQ ID                               NO:109);                                                                     - peptide 237:  LDDLLQKWAEAFNQLLKK       (SEQ ID                               NO:237);                                                                     - peptide 238:  EWLKAFYEKVLEKLKELF*      (SEQ ID                               NO:238);                                                                     - peptide 241:  DWFKAFYDKVFEKFKEFF       (SEQ ID                               NO:241);                                                                     - peptide 242:  GIKKFLGSIWKFIKAFVG       (SEQ ID                               NO:242);                                                                     - peptide 243:  DWFKAFYDKVAEKFKEAF       (SEQ ID                               NO:243);                                                                     - peptide 244:  DWLKAFYDKVAEKLKEAF       (SEQ ID                               NO:244);                                                                     - peptide 245:  DWLKAFYDKVFEKFKEFF       (SEQ ID                               NO:245);                                                                     - peptide 246:  EWLEAFYKKVLEKLKELF       (SEQ ID                               NO:246);                                                                     - peptide 247:  DWFKAFYDKFFEKFKEFF       (SEQ ID                               NO:247);                                                                     - peptide 248:  EWLKAFYEKVLEKLKELF       (SEQ ID                               NO:248);                                                                     - peptide 249:  EWLKAEYEKVEEKLKELF*      (SEQ ID                               NO:249);                                                                     - peptide 250:  EWLKAEYEKVLEKLKELF*      (SEQ ID                               NO:250);                                                                     -   and                                                                       - peptide 251:  EWLKAFYKKVLEKLKELF*      (SEQ ID                               NO:251).                                                                  ______________________________________                                    

In a final preferred embodiment, the ApoA-I agonists are not any of thepeptides listed in TABLE X (Section 8.3, infra) exhibiting an LCATactivation activity of less than 38% as compared with native humanApoA-I.

5.2 SYNTHESIS AND PURIFICATION OF THE ApoA-I PEPTIDE AGONISTS

The core peptides of the invention may be prepared using virtually anyart-known technique for the preparation of peptides. For example, thepeptides may be prepared using conventional step-wise solution or solidphase peptide syntheses, or recombinant DNA techniques.

5.2.1 CHEMICAL SYNTHESIS

Core peptides may be prepared using conventional step-wise solution orsolid phase synthesis (see, e.g., Chemical Approaches to the Synthesisof Peptides and Proteins, Williams et al., Eds., 1997, CRC Press, BocaRaton Fla., and references cited therein; Solid Phase Peptide Synthesis:A Practical Approach, Atherton & Sheppard, Eds., 1989, IRL Press,Oxford, England, and references cited therein).

Alternatively, the peptides of the invention may be prepared by way ofsegment condensation, as described, for example, in Liu et al., 1996,Tetrahedron Lett. 37(7):933-936; Baca, et al., 1995, J. Am. Chem. Soc.117:1881-1887; Tam et al., 1995, Int. J. Peptide Protein Res.45:209-216; Schnolzer and Kent, 1992, Science 256:221-225; Liu and Tam,1994, J. Am. Chem. Soc. 116(10):4149-4153; Liu and Tam, 1994, Proc.Natl. Acad. Sci. USA 91:6584-6588; Yamashiro and Li, 1988, Int. J.Peptide Protein Res. 31:322-334). This is particularly the case withglycine containing peptides. Other methods useful for synthesizing thepeptides of the invention are described in Nakagawa et al., 1985, J. Am.Chem. Soc. 107:7087-7092.

ApoA-I agonists containing N- and/or C-terminal blocking groups can beprepared using standard techniques of organic chemistry. For example,methods for acylating the N-terminus of a peptide or amidating oresterifying the C-terminus of a peptide are well-known in the art. Modesof carrying other modifications at the N- and/or C-terminus will beapparent to those of skill in the art, as will modes of protecting anyside-chain functionalities as may be necessary to attach terminalblocking groups.

Pharmaceutically acceptable salts (counter ions) can be convenientlyprepared by ion-exchange chromatography or other methods as are wellknown in the art.

Compounds of the invention which are in the form of tandem multimers canbe conveniently synthesized by adding the linker(s) to the peptide chainat the appropriate step in the synthesis. Alternatively, the helicalsegments can be synthesized and each segment reacted with the linker. Ofcourse, the actual method of synthesis will depend on the composition ofthe linker. Suitable protecting schemes and chemistries are well known,and will be apparent to those of skill in the art.

Compounds of the invention which are in the form of branched networkscan be conveniently synthesized using the trimeric and tetrameric resinsand chemistries described in Tam, 1988, Proc. Natl. Acad. Sci. USA85:5409-5413 and Demoor et al., 1996, Eur. J. Biochem. 239:74-84.Modifying the synthetic resins and strategies to synthesize branchednetworks of higher or lower order, or which contain combinations ofdifferent core peptide helical segments, is well within the capabilitiesof those of skill in the art of peptide chemistry and/or organicchemistry.

Formation of disulfide linkages, if desired, is generally conducted inthe presence of mild oxidizing agents. Chemical oxidizing agents may beused, or the compounds may simply be exposed to atmospheric oxygen toeffect these linkages. Various methods are known in the art, includingthose described, for example, by Tam et al., 1979, Synthesis 955-957;Stewart et al., 1984, Solid Phase Peptide Synthesis, 2d Ed., PierceChemical Company Rockford, Ill.; Ahmed et al., 1975, J. Biol. Chem.250:8477-8482; and Pennington et al., 1991 Peptides 1990 164-166, Giraltand Andreu, Eds., ESCOM Leiden, The Netherlands. An additionalalternative is described by Kamber et al., 1980, Helv. Chim. Acta63:899-915. A method conducted on solid supports is described byAlbericio, 1985, Int. J. Peptide Protein Res. 26:92-97. Any of thesemethods may be used to form disulfide linkages in the peptides of theinvention.

5.2.2 RECOMBINANT SYNTHESIS

If the peptide is composed entirely of gene-encoded amino acids, or aportion of it is so composed, the peptide or the relevant portion mayalso be synthesized using conventional recombinant genetic engineeringtechniques.

For recombinant production, a polynucleotide sequence encoding thepeptide is inserted into an appropriate expression vehicle, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation.The expression vehicle is then transfected into a suitable target cellwhich will express the peptide. Depending on the expression system used,the expressed peptide is then isolated by procedures well-established inthe art. Methods for recombinant protein and peptide production are wellknown in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning ALaboratory Manual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel etal., 1989, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, N.Y. each of which is incorporated byreference herein in its entirety.)

To increase efficiency of production, the polynucleotide can be designedto encode multiple units of the peptide separated by enzymatic cleavagesites--either homopolymers (repeating peptide units) or heteropolymers(different peptides strung together) can be engineered in this way. Theresulting polypeptide can be cleaved (e.g., by treatment with theappropriate enzyme) in order to recover the peptide units. This canincrease the yield of peptides driven by a single promoter. In apreferred embodiment, a polycistronic polynucleotide can be designed sothat a single mRNA is transcribed which encodes multiple peptides (i.e.,homopolymers or heteropolymers) each coding region operatively linked toa cap-independent translation control sequence; e.g., an internalribosome entry site (IRES). When used in appropriate viral expressionsystems, the translation of each peptide encoded by the mRNA is directedinternally in the transcript; e.g., by the IRES. Thus, the polycistronicconstruct directs the transcription of a single, large polycistronicmRNA which, in turn, directs the translation of multiple, individualpeptides. This approach eliminates the production and enzymaticprocessing of polyproteins and may significantly increase yield ofpeptide driven by a single promoter.

A variety of host-expression vector systems may be utilized to expressthe peptides described herein. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used in the expressionvector. For example, when cloning in bacterial systems, induciblepromoters such as pL of bacteriophage λ, plac, ptrp, ptac (ptrp-lachybrid promoter) and the like may be used; when cloning in insect cellsystems, promoters such as the baculovirus polyhedron promoter may beused; when cloning in plant cell systems, promoters derived from thegenome of plant cells (e.g., heat shock promoters; the promoter for thesmall subunit of RUBISCO; the promoter for the chlorophyll a/b bindingprotein) or from plant viruses (e.g., the 35S RNA promoter of CaMV; thecoat protein promoter of TMV) may be used; when cloning in mammaliancell systems, promoters derived from the genome of mammalian cells(e.g., metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5 K promoter) may beused; when generating cell lines that contain multiple copies ofexpression product, SV40-, BPV- and EBV-based vectors may be used withan appropriate selectable marker.

In cases where plant expression vectors are used, the expression ofsequences encoding the peptides of the invention may be driven by any ofa number of promoters. For example, viral promoters such as the 35S RNAand 19S RNA promoters of CaMV (Brisson et al., 1984, Nature310:511-514), or the coat protein promoter of TMV (Takamatsu et al.,1987, EMBO J. 6:307-311) may be used; alternatively, plant promoterssuch as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J.3:1671-1680; Broglie et al., 1984, Science 224:838-843) or heat shockpromoters, e.g., soybean hsp17.5-E or hsp17.3-B (Gurley et al., 1986,Mol. Cell. Biol. 6:559-565) may be used. These constructs can beintroduced into plant cells using Ti plasmids, Ri plasmids, plant virusvectors, direct DNA transformation, microinjection, electroporation,etc. For reviews of such techniques see, e.g., Weissbach & Weissbach,1988, Methods for Plant Molecular Biology, Academic Press, N.Y., SectionVIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology,2d Ed., Blackie, London, Ch. 7-9.

In one insect expression system that may be used to produce the peptidesof the invention, Autographa californica, nuclear polyhidrosis virus(AcNPV) is used as a vector to express the foreign genes. The virusgrows in Spodoptera frugiperda cells. A coding sequence may be clonedinto non-essential regions (for example the polyhedron gene) of thevirus and placed under control of an AcNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed (e.g., see Smith et al., 1983, J. Virol. 46:584;Smith, U.S. Pat. No. 4,215,051). Further examples of this expressionsystem may be found in Current Protocols in Molecular Biology, Vol. 2,Ausubel et al., eds., Greene Publish. Assoc. & Wiley Interscience.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. (e.g., See Logan & Shenk, 1984, Proc. Natl.Acad. Sci. (USA) 81:3655-3659). Alternatively, the vaccinia 7.5 Kpromoter may be used, (see, e.g., Mackett et al., 1982, Proc. Natl.Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984, J. Virol.49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci. 79:4927-4931).

Other expression systems for producing the peptides of the inventionwill be apparent to those having skill in the art.

5.2.3 PURIFICATION OF PEPTIDES

The peptides of the invention can be purified by art-known techniquessuch as reverse phase chromatography high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography and the like. The actual conditions used topurify a particular peptide will depend, in part, on synthesis strategyand on factors such as net charge, hydrophobicity, hydrophilicity, etc.,and will be apparent to those having skill in the art. Multimericbranched peptides can be purified, e.g., by ion exchange or sizeexclusion chromatography.

For affinity chromatography purification, any antibody whichspecifically binds the peptide may be used. For the production ofantibodies, various host animals, including but not limited to rabbits,mice, rats, etc., may be immunized by injection with a peptide. Thepeptide may be attached to a suitable carrier, such as BSA, by means ofa side chain functional group or linkers attached to a side chainfunctional group. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies to a peptide may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include but are not limited to thehybridoma technique originally described by Kohler and Milstein, 1975,Nature 256:495-497, or Kaprowski, U.S. Pat. No. 4,376,110 which isincorporated by reference herein; the human B-cell hybridoma technique)Kosbor et al., 1983, Immunology Today 4:72; Cote et al., 1983, Proc.Natl. Acad. Sci. U.S.A. 80:2026-2030); and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96 (1985)). In addition, techniques developed for theproduction of "chimeric antibodies" Morrison et al., 1984, Proc. Natl.Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature312:604-608; Takeda et al., 1985, Nature 314:452-454, Boss, U.S. Pat.No. 4,816,397; Cabilly, U.S. Pat. No. 4,816,567; which are incorporatedby reference herein) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.Or "humanized" antibodies can be prepared (see, e.g., Queen, U.S. Pat.No. 5,585,089 which is incorporated by reference herein). Alternatively,techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce peptide-specific singlechain antibodies.

Antibody fragments which contain deletions of specific binding sites maybe generated by known techniques. For example, such fragments includebut are not limited to F(ab')₂ fragments, which can be produced bypepsin digestion of the antibody molecule and Fab fragments, which canbe generated by reducing the disulfide bridges of the F(ab')₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for the peptide ofinterest.

The antibody or antibody fragment specific for the desired peptide canbe attached, for example, to agarose, and the antibody-agarose complexis used in immunochromatography to purify peptides of the invention.See, Scopes, 1984, Protein Purification: Principles and Practice,Springer-Verlag New York, Inc., N.Y., Livingstone, 1974, Methods InEnzymology: Immunoaffinity Chromatography of Proteins 34:723-731.

5.3 PHARMACEUTICAL FORMULATIONS AND METHODS OF TREATMENT

The ApoA-I agonists of the invention can be used to treat any disorderin animals, especially mammals including humans, for which increasingserum HDL concentration, activating LCAT, and promoting cholesterolefflux and RCT is beneficial. Such conditions include, but are notlimited to hyperlipidemia, and especially hypercholesterolemia, andcardiovascular disease such as atherosclerosis (including treatment andprevention of atherosclerosis); restenosis (e.g., preventing or treatingatherosclerotic plaques which develop as a consequence of medicalprocedures such as balloon angioplasty); and other disorders, such asendotoxemia, which often results in septic shock.

The ApoA-I agonists can be used alone or in combination therapy withother drugs used to treat the foregoing conditions. Such therapiesinclude, but are not limited to simultaneous or sequentialadministration of the drugs involved.

For example, in the treatment of hypercholesterolemia oratherosclerosis, the ApoA-I agonist formulations can be administeredwith any one or more of the cholesterol lowering therapies currently inuse; e.g., bile-acid resins, niacin, and/or statins. Such a combinedregimen may produce particularly beneficial therapeutic effects sinceeach drug acts on a different target in cholesterol synthesis andtransport; i.e., bile-acid resins affect cholesterol recycling, thechylomicron and LDL population; niacin primarily affects the VLDL andLDL population; the statins inhibit cholesterol synthesis, decreasingthe LDL population (and perhaps increasing LDL receptor expression);whereas the ApoA-I agonists affect RCT, increase HDL, increase LCATactivity and promote cholesterol efflux.

In another embodiment, the ApoA-I agonists may be used in conjunctionwith fibrates to treat hyperlipidemia, hypercholesterolemia and/orcardiovascular disease such as atherosclerosis.

In yet another embodiment, the ApoA-I agonists of the invention can beused in combination with the anti-microbials and anti-inflammatoryagents currently used to treat septic shock induced by endotoxin.

The ApoA-I agonists of the invention can be formulated as peptides or aspeptide-lipid complexes which can be administered to subjects in avariety of ways to deliver the ApoA-l agonist to the circulation.Exemplary formulations and treatment regimens are described below.

5.3.1 ApoA-I AGONISTS AND PEPTIDE/LIPID COMPLEX AS THE ACTIVE INGREDIENT

The ApoA-I agonist peptides can be synthesized or manufactured using anytechnique described in Section 5.2 and its subsections. Stablepreparations which have a long shelf life may be made by lyophilizingthe peptides--either to prepare bulk for reformulation, or to prepareindividual aliquots or dosage units which can be reconstituted byrehydration with sterile water or an appropriate sterile bufferedsolution prior to administration to a subject.

In certain embodiments, it may be preferred to formulate and administerthe ApoA-I agonist in a peptide-lipid complex. This approach has severaladvantages since the complex should have an increased half-life in thecirculation, particularly when the complex has a similar size anddensity to HDL, and especially the pre-β-1 or pre-β-2 HDL populations.The peptide-lipid complexes can conveniently be prepared by any of anumber of methods described below. Stable preparations having a longshelf life may be made by lyophilization--the co-lyophilizationprocedure described below being the preferred approach. The lyophilizedpeptide-lipid complexes can be used to prepare bulk for pharmaceuticalreformulation, or to prepare individual aliquots or dosage units whichcan be reconstituted by rehydration with sterile water or an appropriatebuffered solution prior to administration to a subject.

A variety of methods well known to those skilled in the art can be usedto prepare the peptide-lipid vesicles or complexes. To this end, anumber of available techniques for preparing liposomes orproteoliposomes may be used. For example, the peptide can be cosonicated(using a bath or probe sonicator) with appropriate lipids to formcomplexes. Alternatively the peptide can be combined with preformedlipid vesicles resulting in the spontaneous formation of peptide-lipidcomplexes. In yet another alternative, the peptide-lipid complexes canbe formed by a detergent dialysis method; e.g., a mixture of thepeptide, lipid and detergent is dialyzed to remove the detergent andreconstitute or form peptide-lipid complexes (e.g., see Jonas et al.,1986, Methods in Enzymol. 128:553-582).

While the foregoing approaches are feasible, each method presents itsown peculiar production problems in terms of cost, yield,reproducibility and safety. The applicants have developed a simplemethod for preparing peptide or protein-phospholipid complexes whichhave characteristics similar to HDL. This method can be used to preparethe ApoA-I peptide-lipid complexes, and has the following advantages:(1) Most or all of the included ingredients are used to form thedesigned complexes, thus avoiding waste of starting material which iscommon to the other methods. (2) Lyophilized compounds are formed whichare very stable during storage. The resulting complexes may bereconstituted immediately before use. (3) The resulting complexesusually need not be further purified after formation and before use. (4)Toxic compounds, including detergents such as cholate, are avoided.Moreover, the production method can be easily scaled up and is suitablefor GMP manufacture (i.e., in an endotoxin-free environment).

In accordance with the preferred method, the peptide and lipid arecombined in a solvent system which co-solubilizes each ingredient andcan be completely removed by lyophilization. To this end, solvent pairsmust be carefully selected to ensure co-solubility of both theamphipathic peptide and the lipid. In one embodiment, the protein(s) orpeptide(s) to be incorporated into the particles can be dissolved in anaqueous or organic solvent or mixture of solvents (solvent 1). The(phospho)lipid component is dissolved in an aqueous or organic solventor mixture of solvents (solvent 2) which is miscible with solvent 1, andthe two solutions are mixed. Alternatively, the peptide and lipid can beincorporated into a co-solvent system; i.e., a mixture of the misciblesolvents. A suitable proportion of peptide (protein) to lipids is firstdetermined empirically so that the resulting complexes possess theappropriate physical and chemical properties; i.e., usually (but notnecessarily) similar in size to HDL. The resulting mixture is frozen andlyophilized to dryness. Sometimes an additional solvent must be added tothe mixture to facilitate lyophilization. This lyophilized product canbe stored for long periods and will remain stable.

In the working examples describe infra, peptide 4 (SEQ ID NO:4) andphospholipids were dissolved separately in methanol, combined, thenmixed with xylene before lyophilization. The peptide and lipid can bothbe added to a mixture of the two solvents. Alternatively, a solution ofthe peptide dissolved in methanol can be mixed with a solution of lipiddissolved in xylene. Care should be taken to eliminate salt from thesolvent system in order to avoid salting out the peptide. The resultingsolution containing the peptide and lipid cosolubilized inmethanol/xylene is lyophilized to form a powder.

The lyophilized product can be reconstituted in order to obtain asolution or suspension of the peptide-lipid complex. To this end, thelyophilized powder is rehydrated with an aqueous solution to a suitablevolume (often 5 mgs peptide/ml which is convenient for intravenousinjection). In a preferred embodiment the lyophilized powder isrehydrated with phosphate buffered saline or a physiological salinesolution. The mixture may have to be agitated or vortexed to facilitaterehydration, and in most cases, the reconstitution step should beconducted at a temperature equal to or greater than the phase transitiontemperature of the lipid component of the complexes. Within minutes, aclear preparation of reconstituted lipid-protein complexes results.

An aliquot of the resulting reconstituted preparation can becharacterized to confirm that the complexes in the preparation have thedesired size distribution; e.g., the size distribution of HDL. Gelfiltration chromatography can be used to this end. In the workingexamples described infra, a Pharmacia Superose 6 FPLC gel filtrationchromatography system was used. The buffer used contains 150 mM NaCl in50 mM phosphate buffer, pH 7.4. A typical sample volume is 20 to 200microliters of complexes containing 5 mgs peptide/ml. The column flowrate is 0.5 mls/min. A series of proteins of known molecular weight andStokes' diameter as well as human HDL are used as standards to calibratethe column. The proteins and lipoprotein complexes are monitored byabsorbance or scattering of light of wavelength 254 or 280 nm.

The ApoA-I agonists of the invention can be complexed with a variety oflipids, including saturated, unsaturated, natural and synthetic lipidsand/or phospholipids. Suitable lipids include, but are not limited to,small alkyl chain phospholipids, egg phosphatidylcholine, soybeanphosphatidylcholine, dipalmitoylphosphatidylcholine,dimyristoylphosphatidylcholine, distearoylphosphatidylcholine1-myristoyl-2-palmitoylphosphatidylcholine,1-palmitoyl-2-myristoylphosphatidylcholine,1-palmitoyl-2-stearoylphosphatidylcholine,1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholinedioleophosphatidylethanolamine, dilauroylphosphatidylglycerolphosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,phosphatidylinositol, sphingomyelin, sphingolipids,phosphatidylglycerol, diphosphatidylglycerol,dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol,distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol,dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid,dimyristoylphosphatidylethanolamine,dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine,dipalmitoylphosphatidylserine, brain phosphatidylserine, brainsphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin,phosphatidic acid, galactocerebroside, gangliosides, cerebrosides,dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride,aminophenylglycoside, 3-cholesteryl-6'-(glycosylthio)hexyl etherglycolipids, and cholesterol and its derivatives.

The Applicants have discovered that when the ApoA-I agonists of theinvention are complexed with sphingomyelin, all of the HDL of thepre-β-like particles is removed. Accordingly, in a preferred embodimentof the invention, the ApoA-I agonists are administered as a complex withsphingomyelin.

5.3.2 METHODS OF THE TREATMENT

The ApoA-I peptide agonists or peptide-lipid complexes of the inventionmay be administered by any suitable route that ensures bioavailabilityin the circulation. This can best be achieved by parenteral routes ofadministration, including intravenous (IV), intramuscular (IM),intradermal, subcutaneous (SC) and intraperitoneal (IP) injections.However, other routes of administration may be used. For example,absorption through the gastrointestinal tract can be accomplished byoral routes of administration (including but not limited to ingestion,buccal and sublingual routes) provided appropriate formulations (e.g.,enteric coatings) are used to avoid or minimize degradation of theactive ingredient, e.g., in the harsh environments of the oral mucosa,stomach and/or small intestine. Alternatively, administration viamucosal tissue such as vaginal and rectal modes of administration may beutilized to avoid or minimize degradation in the gastrointestinal tract.In yet another alternative, the formulations of the invention can beadministered transcutaneously (e.g., transdermally), or by inhalation.It will be appreciated that the preferred route may vary with thecondition, age and compliance of the recipient.

The actual dose of ApoA-I agonists or peptide-lipid complex used willvary with the route of administration, and should be adjusted to achievecirculating plasma concentrations of 100 mg/l to 2 g/l. Data obtained inanimal model systems described herein show that the ApoA-I agonists ofthe invention associate with the HDL component, and have a projectedhalf-life in humans of about five days. Thus, in one embodiment, theApoA-I agonists can be administered by injection at a dose between 0.5mg/kg to 100 mg/kg once a week. In another embodiment, desirable serumlevels may be maintained by continuous infusion or by intermittentinfusion providing about 0.5 mg/kg/hr to 100 mg/kg/hr.

Toxicity and therapeutic efficacy of the various ApoA-I agonists can bedetermined using standard pharmaceutical procedures in cell culture orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. ApoA-I peptide agonists which exhibit large therapeutic indicesare preferred.

5.3.3 PHARMACEUTICAL FORMULATIONS

The pharmaceutical formulation of the invention contain the ApoA-Ipeptide agonist or the peptide-lipid complex as the active ingredient ina pharmaceutically acceptable carrier suitable for administration anddelivery in vivo. As the peptides may contain acidic and/or basictermini and/or side chains, the peptides can be included in theformulations in either the form of free acids or bases, or in the formof pharmaceutically acceptable salts.

Injectable preparations include sterile suspensions, solutions oremulsions of the active ingredient in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives.

Alternatively, the injectable formulation may be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.To this end, the ApoA-I agonist may be lyophilized, or theco-lyophilized peptide-lipid complex may be prepared. The storedpreparations can be supplied in unit dosage forms and reconstitutedprior to use in vivo.

For prolonged delivery, the active ingredient can be formulated as adepot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt form of the ApoA-I agonist.

Alternatively, transdermal delivery systems manufactured as an adhesivedisc or patch which slowly releases the active ingredient forpercutaneous absorption may be used. To this end, permeation enhancersmay be used to facilitate transdermal penetration of the activeingredient. A particular benefit may be achieved by incorporating theApoA-I agonists of the invention or the peptide-lipid complex into anitroglycerin patch for use in patients with ischemic heart disease andhypercholesterolemia.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner. For rectal and vaginalroutes of administration, the active ingredient may be formulated assolutions (for retention enemas) suppositories or ointments.

For administration by inhalation, the active ingredient can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

5.4 OTHER USES

The ApoA-I agonists of the invention can be used in assays in vitro tomeasure serum HDL, e.g., for diagnostic purposes. Because the ApoA-Iagonists associate with the HDL component of serum, the agonists can beused as "markers" for the HDL population. Moreover, the agonists can beused as markers for the subpopulation of HDL that are effective in RCT.To this end, the agonist can be added to or mixed with a patient serumsample; after an appropriate incubation time, the HDL component can beassayed by detecting the incorporated ApoA-I agonist. This can beaccomplished using labeled agonist (e.g., radiolabels, fluorescentlabels, enzyme labels, dyes, etc.), or by immunoassays using antibodies(or antibody fragments) specific for the agonist.

Alternatively, labeled agonist can be used in imaging procedures (e.g.,CAT scans, MRI scans) to visualize the circulatory system, or to monitorRCT, or to visualize accumulation of HDL at fatty streaks,atherosclerotic lesions, etc. (where the HDL should be active incholesterol efflux).

6. EXAMPLE: SYNTHESIS OF PEPTIDE AGONISTS OF ApoA-I

The peptides described in TABLE X (Section 8.3, infra) were synthesizedand characterized as described in the subsections below. The peptideswere also analyzed structurally and functionally as described inSections 7 and 8, infra.

6.1 SYNTHESIS OF CORE PEPTIDES

Peptides were synthesized on solid phase according to the Merrifieldtechnique (Merrifield, 1969, J. Am. Chem. Soc. 85:2149-2154) using 0.25mmol ρ-alkoxybenzylalcohol resin (HMP resin) (Wang, 1973, J. Am. Chem.Soc. 95:1328-1333) and Fmoc chemistry. All syntheses were carried out onan Applied Biosystems ABI model 430A automated peptide synthesizer(Perkin-Elmer, Foster City, Calif.). The solvation and activation timesused for each coupling cycle are shown in TABLE V below:

                                      TABLE V                                     __________________________________________________________________________    SINGLE COUPLE ACTIVATOR CYCLES                                                CYCLE                                                                             DESIGNATED                                                                            DISSOLVING   ACTIVATION                                                                           TRANSFER                                        NAME AMINO ACIDS SOLVENT TIME TIME TIMES*                                   __________________________________________________________________________    afmc 31                                                                           Asn(trt),                                                                             ˜0.4 ml DCM                                                                     ˜7 min.                                                                      ˜51 min.                                                                       1 = 50 sec.                                      His(trt), ˜1.2 ml NMP   2 = 36 sec.                                     Lys(Boc),Trp ˜1.0 ml                                                     HOBt/NMP                                                                    afmc 32 Arg(Pmc), ˜0.8 ml DCM ˜32 min. ˜51 min. 1 =                                       60 sec.                                          Gln(trt),Aib ˜1.2 ml NMP   2 = 40 sec.                                   ˜1.0 ml                                                                 HOBt/NMP                                                                    afmc 33 Ala,Asp(otBu), ˜0.4 ml DCM  ˜4 min. ˜36.5 1 =                                     38 sec.                                          Glu(OtBu),Gly, ˜0.8 ml NMP  min. 2 = 27 sec.                            Ile,Leu, ˜0.1 ml                                                        Met, Phe, Pro HOBt/NMP                                                       afmc 34 Val ˜0.4 ml DCM  ˜4 min. ˜61.5 1 = 38 sec.                                           ˜0.8 ml NMP  min. 2 = 27 sec.                                           ˜0.1 ml                                  HOBt/NMP                                                                  __________________________________________________________________________     *1 = Transfer from Cartridge to Activator.                                    2 = Transfer from Activator to Cartridge.                                     DCC is dicyclohexylcarbodiimide                                               BOC is tbutyloxycarbonyl                                                      HOBt is 1hydroxybenzotriazole                                                 Pmc is pentamethylchroman6-sulfonyl                                           NMP is Nmethylpyrrolidone                                                     OtBu is tbutyl ester                                                          trt is trityl                                                            

The resins were washed with NMP between each coupling step. The protocolfor one synthesis cycle is shown below in TABLE VI:

                  TABLE VI                                                        ______________________________________                                        COUPLING PROTOCOL FOR ONE SYNTHESIS CYCLE                                              OPERATION        TIME (min.)                                         ______________________________________                                        1.       Deprotection (10% piperdine                                                                    20                                                     in NMP)                                                                      2. Wash (NMP)  5                                                              3. Couple (4 equiv. Fmoc-amino 61                                              acid-HOBT ester in NMP,                                                       preactivated 50 min.)                                                        4. Wash  3                                                                    5. Resin Sample (optional)  3                                                  TOTAL 92                                                                   ______________________________________                                    

All amino acids except Fmoc-β-(1-naphthyl)alanine were coupled in thismanner. Fmoc-β-(1-naphthyl)alanine was coupled manually. For manualcoupling, 1 mmol Fmoc-β-(1-naphthyl)alanine and 1 mmol2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) were dissolved in 5 ml NMP and mixed with the peptide-resin.

Thereafter, 2 mmol of N-ethyldiisopropylamine were added, the mixtureshaken for 2 hours and the peptide-resin washed 6 times with 10 ml NMP.The coupling efficiency was monitored using the Kaiser Test (Kaiser,1970, Anal. Biochem. 34:59577), and the coupling repeated if necessary.After coupling of naphthylalanine, the remainder of the synthesis wasperformed automatically as described above.

6.2 SYNTHESIS OF PEPTIDE AMIDES

Where indicated in TABLE X (Section 8.3, infra), peptide amides weresynthesized using a Rink amide resin containing the Fmoc-Rink amidehandle 4-(2',4'-dimethylphenyl)-Fmoc-phenoxymethyl (Rink, 1987,Tetrahedron Lett. 28:3787-3790) and the synthesis protocols described inSection 6.1, supra.

6.3 SYNTHESIS OF N-TERMINAL ACYLATED PEPTIDES

Where indicated in TABLE X (Section 8.3, infra), N-terminal acylatedforms of the peptides were prepared by exposing the resin-bound peptideprepared as described in Section 6.1 or 6.2, supra, to an appropriateacylating agent.

For N-terminal acetylated peptides, 15 ml of acetic anhydride solution(10% v/v in NMP) was added to each 1 g of resin-bound peptide, themixture shaken for 5 min. and the resin recovered by filtration. Therecovered resin was washed three times with NMP (15 ml) and three timeswith ethanol (15 ml).

6.4 CLEAVAGE AND DEPROTECTION

Following synthesis, the peptides described in Sections 6.1, 6.2 and6.3, supra, were cleaved from the resin and deprotected with a cleavagesolution containing 92.5% trifluroacetic acid (TFA)/3.75% anisole/3.75%dodecanthiol (v/v/v). To effect cleavage, 10 ml of cleavage solution wasadded to 0.25 mmol peptide resin and stirred for 1.5 hours at roomtemperature. The resin was removed via filtration and thecleaved/deprotected peptide precipitated with diethyl ether, washed withether and dried in vacuo.

The cleavage cocktail for peptides containing Trp (W), as well as forpeptide amides, was composed of 86.5% TFA, 4.5% H₂ O, 4.5%1,2-ethanedithiol, 4.5% anisole and 3% phenol.

6.5 PURIFICATION

The crude, cleaved peptides of Section 6.4 were purified by reversephase HPLC. The purity of each peptide was confirmed by differentanalytical techniques (analytical HPLC, capillary electrophoresis).Capillary electrophoreses were carried out on fused silica capillariesof 70 cm length and an internal diameter of 75 μm (Thermo SeparationProducts). The separations were performed at 250C, 15 kV, run time 35min., in two different buffer systems: Buffer 1 (20 mM Na₂ B₄ O₇, pH9.2) and Buffer 2 (10 mM Na₂ HPO₄, pH 2.5). HPLC separations werecarried out on Nucleosil 7C18 or Nucleosil 7C4 columns (Macherey andNagel, Germany), 250×21 mm, at a flow rate of 8 ml/min. The gradientelution was performed using a mixture of 0.1% TFA in water (Solvent A)and 0.1% TFA in acetonitrile (Solvent B). The gradients used wereadjusted to meet the needs of each peptide.

6.6 CHARACTERIZATION

The mass and amino acid analysis of the purified peptides described inSection 6.5 were confirmed via mass spectrometry and amino acidanalysis, respectively, as described below. Edman degradation was usedfor sequencing.

6.6.1 LC-MS

A standard commercially available triple stage quadruple massspectrometer (model TSQ 700; Finnigan MAT, San Jose Calif., USA) wasused for mass determination. A pneumatically assisted electrospray (ESI)interface was used for sample introduction to the atmospheric pressureionization source of the mass spectrometer. The interface sprayer wasoperated at a positive potential of 4.5 kV. The temperature of the steelcapillary was held at 200° C. whereas the manifold was at 70° C.Positive ions generated by this ion evaporation process entered theanalyzer of the mass spectrometer. The multiplier was adjusted to 1000V. The analyzer compartment of the mass spectrometer was at 4E-6. Allacquisitions were performed at resolution <1 u.

Peptides were analyzed by direct infusion of the purified peptides usingan ABI (Applied Biosystems) microbore system consisting of a syringepump (model 140B), an UV detector (model 785A) and an oven/injector(model 112A). The solvent system consisted of water (solvent A) andacetonitrile (solvent B), each containing 0.1% TFA. Peptides wereinfused using either a gradient or isocratic conditions and eluted froman Aquapore C18 column. The flow rate was typically 300 μl/min.Concentration of each peptide was about 0.03 mg/ml, 20 μl of which wasinjected (e.g., 30 pmol).

Full scan MS experiments were obtained by scanning quadruple 1 from m/z500-1500 in 4 s. Data were acquired using an Alpha DEC station and wereprocessed using the software package provided by Finnigan MAT(BIOWORKS).

6.6.2 AMINO ACID ANALYSIS

Amino acid analysis was performed on an ABI (Applied Biosystems) 420Amino Acid Analyzer. This system consists of three modules: a hydrolysisand derivatisation instrument, a reverse-phase HPLC and a data system.Peptide sample were applied (3 times in triplicate) on porous glassslides and subsequently hydrolyzed under gas phase conditions (155° C.,90 min.). After removal of the HCL, the resulting amino acids wereconverted to PTC-AA (Phenylthiocarbamoyl-amino acids) using PITC(Phenylisothiocyanate). After transfer to the HPC sample loop theresulting mixtures were fractionated on an Aquapore C18 column using thegradient mode (Solvent A: 50 mmol ammonium acetate (NH₄ Ac), pH 5.4, inwater; Solvent B: 32 mmol of sodium acetate (NaOAc) in aqueousacetonitrile) under conditions of temperature control. The HPLC datawere processed by the software package provided by Applied Biosystems.Quantification was performed relative to a peptide standard delivered byApplied Biosystems.

6.7 SYNTHESIS OF BRANCHED NETWORKS

Tetrameric-core peptidyl resin and trimeric-core peptidyl resin aresynthesized as described in Demoor et al., 1996, Eur. J. Biochem.239:74-84. The tetrameric and trimeric core matrix still linked to the4-methyl benzhydrylamine resin is then used as initial peptidyl-resinfor automated synthesis of core peptides as previously described.

Branched networks containing helical segments of different amino acidcompositions can be synthesized using orthogonal synthesis andprotecting strategies well known in the art.

7. EXAMPLE: STRUCTURAL AND LIPID BINDING ANALYSIS OF ApoA-I PEPTIDES

The structural and lipid binding characteristics of the purifiedpeptides synthesized as described in Section 6, supra, were determinedby circular dichroism (CD), fluorescence spectroscopy and nuclearmagnetic resonance (NMR).

7.1 CIRCULAR DICHROISM

This Example describes a preferred method for determining the degree ofhelicity of the core peptides of the invention both free in buffer andin the presence of lipids.

7.1.1 EXPERIMENTAL METHOD

Far UV circular dichroism spectra were recorded between 190 and 260 nm(in 0.5 nm or 0.2 nm increments) with a AVIV62DS spectrometer (AVIVAssociates, Lakewood, N.J., USA) equipped with a thermoelectric cellholder and sample changer. The instrument was calibrated with(+)-10-camphoric acid. Between one and three scans were collected foreach sample, using 10 cm, 5 cm, 1 cm and 0.1 cm path length quartzSuprasil cells, respectively, for peptide concentrations of 10⁻⁷ M to10⁻⁴ M. The bandwidth was fixed at 1.5 nm and the scan speed to 1 s perwavelength step. The reported data are the mean of at least 2 or 3independent measurements.

After background substraction, spectra were converted to molarellipticity (θ) per residue in deg. cm⁻² dmol⁻¹. The peptideconcentration was determined by amino acid analysis and also byabsorption spectrometry on a Perkin Elmer Lambda 17 UV/Visiblespectrophotometer when the peptide contained a chromophore (tryptophane,dansyl, naphtylalanine).

CD spectra were obtained with free, unbound peptide (5 μM in 5 mMphosphate buffer, pH 7.4); with peptide-SUV complexes (20:1 EPC:Chol.,Ri=30 and Ri=50); with peptide-micelle complexes(1-myristoyl-2-hydroxy-sn-glycero-3-phosphatidyl choline, Ri=100); andwith free, unbound peptide in the presence of 2,2,2-trifluoroethanol(TFE) (5 μM peptide, 90% vol TFE).

The SUVs were obtained by dispersing the lipids (10 mM, 20:1 EPC:Chol.,Avanti Polar Lipids, AL) in phosphate buffer (5 mM, pH 7.4) withbubbling N₂ for 5 min., followed by sonication (1.5 hr.) in a bathsonicator. The homogeneity of the preparation was checked by FPLC.

The micelles were obtained by dispersing the lipid (6 mM1-myristoyl-2-hydroxy-sn-glycero-3-phosphatidyl choline, Avanti PolarLipids, AL) in phosphate buffer (5 mM, pH 7.4) with bubbling N₂ for 5min., followed by vortexing.

To obtain the peptide-SUV complexes, SUVs were added to the peptide (5μM in 5 mM phosphate buffer, pH 7.4) at a phospholipid-peptide molarratio (Ri) of 30 or 50.

To obtain the peptide-micelle complexes, micelles were added to thepeptide (5 μM in 5 mM phosphate buffer, pH 7.4) at a Ri of 100.

All spectra were recorded at 37° C. The stability of peptide 4 (SEQ IDNO:4) as a function of temperature (both free in buffer and in micelles)was determined by recording spectra at a series of differenttemperatures.

The degree of helicity of peptide 4 (SEQ ID NO:4) as a function ofconcentration was also determined.

7.1.2 HELICITY DETERMINATION

The degree of helicity of the peptides in the various conditions wasdetermined from the mean residue ellipticity at 222 nm (Chen et al.,1974, Biochemistry 13:3350-3359) or by comparing the CD spectra obtainedto reference spectra available on databases (16 helical referencespectra from Provencher & Glockner, 1981, Biochemistry 20:33-37;denatured protein reference spectra from Venyaminov et al., 1993, Anal.Biochem. 214:17-24) using the CONTIN curve-fitting algorithm version2DP, CD-1 pack (August 1982) (Provencher, 1982, Comput. Phys. Commun.27:213-227, 229-242). Acceptable fit was determined using thestatistical analysis methodology provided by the CONTIN algorithm. Theerror of all methods was ±5% helicity.

7.1.3 RESULTS

The degree of helicity (%) of the free, unbound peptides (free), thepeptide-SUV complexes (SUVs), the peptide-micelle complexes (mics) andthe peptide-TFE solution (TFE) are reported in TABLE X, Section 8.3,infra. The degree of helicity of peptide 4 (SEQ ID NO:4) as a functionof concentration (free and bound to lipids) is provided in TABLE VII.

                  TABLE VII                                                       ______________________________________                                        % HELICITY OF PEPTIDE 4 (SEQ ID NO: 4)                                          AS A FUNCTION OF CONCENTRATION                                                                           % Helicity                                           Bound                                                                       Concentration (μM) Free (micelles) TFE                                   ______________________________________                                        0.15          42         65       --                                            0.40 58 68 --                                                                 2.0 63 -- --                                                                  5.0 80 95 --                                                                  50.0 -- -- 94                                                               ______________________________________                                    

Peptide 4 (SEQ ID NO:4) contains significant α-helical structure (80%helicity) in buffer at a concentration of 5 μM. While the degree ofhelicity decreases with decreasing peptide concentration, significanthelicity (42%) is maintained even at concentrations as low as 0.15 μM.Moreover, the α-helical structure is completely stable over atemperature range of 5-45° C. (data not shown).

The helicity of peptide 4 (SEQ ID NO:4) increases in the presence ofboth SUVs (97% helicity) and micelles (95% helicity), and also in thepresence TFE (94% helicity), which is a solvent that, due to having asignificantly lower dielectric constant (ε=26.7) that water (ε=78.4),stabilizes α-helices and intrapeptide hydrogen bonds at concentrationsbetween 5-90% (v/v).

Referring to TABLE X, Section 8.3, infra, it can be seen that thosepeptides which exhibit a high degree of LCAT activation (≧38%) generallypossess significant α-helical structure in the presence of lipids (≧60%helical structure in the case of unblocked peptides containing 22 ormore amino acids or blocked peptides containing 18 or fewer amino acids;≧40% helical structure in the case of unblocked peptides containing 18or fewer amino acids), whereas peptides which exhibit little or no LCATactivation possess little α-helical structure. However, in someinstances, peptides which contain significant α-helical structure in thepresence of lipids do not exhibit significant LCAT activation. As aconsequence, the ability of the core peptides of the invention to adoptan α-helical structure in the presence of lipids is considered acritical feature of the core peptides of the invention, as the abilityto form an α-helix in the presence of lipids appears to be aprerequisite for LCAT activation.

7.2 FLUORESCENCE SPECTROSCOPY

The lipid binding properties of the peptides synthesized in Section 6,supra, were tested by fluorescence measurements with labeled peptides,in the present case Tryptophane (Trp or W) or Naphtylalanine (Nal). Thefluorescence spectra were recorded on a Fluoromax from Spex (Jobin-Yvon)equipped with a Xenon lamp of 150 W, two monochromators (excitation andemission), a photomultiplier R-928 for detection sensitive in the red upto 850 nm and a thermoelectric magnetic stirred cell holder. QuartzSuprasil cuvettes were used for measurements in the micromolarconcentration range. A device of variable slits (from 0.4 to 5 nm)allows modulation of the incident and emitted intensities according tothe concentration of peptide used. The reported values are in generalthe average of between 2 to 4 spectra. The peptide concentration isdetermined by absorption spectrometry on a Philips PU 8800 using theabsorption band of the Trp (ε₂₈₀ nm =5,550 M⁻¹ cm⁻¹ in Tris buffer) orthe Nal (ε₂₂₄ nm =92,770 M⁻¹ cm⁻¹ in methanol).

Fluorescence spectra of the peptides were recorded between 290 nm and450 nm in Tris-HCl buffer (20 mM, pH=7.5), in the presence and absenceof lipidic vesicles. The small unilamellar vesicles were formed afterrehydration in buffer of the lyophilized phospholipids, dispersion andtip sonification under a N₂ stream. The lipids used were either EggPC/Chol. (20:1) or POPC/Chol. (20:1). The spectra were recorded at apeptide concentration of 2 μM and at a temperature of 37° C. Thefluorescence reference standard in the case of Trp wasN-acetyltryptophanylamide (NATA).

Lipid binding studies were done through progressive lipidic vesicleaddition to the peptide in solution at 2 μM (slits:5 nm in excitationand 1.5 nm in emission). Dilution effects were taken into account forthe fluorescence intensity determination. The lipid concentrations werevaried from 10 to 600 μM and the molar ratio of lipid to peptide (Ri)was varied from 5 to 300. The wavelength of excitation was set at 280 nmfor both Trp and Nal.

7.2.1 FLUORESCENCE SPECTRAL ANALYSIS

The data were directly recorded and treated by an IBM-PC linked to thespectrofluorimeter through the DM3000F software from Spex. The spectrawere corrected by substraction of the solvent contribution and byapplication of a coefficient given by the constructor taking intoaccount the variation of the photomultiplier response versus thewavelength.

The fluorescence spectra of the peptides were characterized by thewavelength at their maximum of fluorescence emission and by theirquantum yield compared to NATA in the case of peptides labeled with atryptophane. The process of binding to lipids was analyzed bycalculating the shift of the wavelength at the maximum of fluorescenceemission, (λ_(max)), and the variation of the relative fluorescenceintensity of emission versus the lipid concentration. The relativefluorescence intensity is defined as the following ratio:(I-I₀).sub.λmax /I₀λmax. I and I₀ are both measured at the (λ_(max))corresponding to the initial free state of the peptide, i.e., withoutlipids. I is the intensity at a defined lipid to peptide ratio and I₀ isthe same parameter measured in absence of lipids. The absence of thesevariations is relevant of the absence of interactions of the peptideswith the lipids.

7.2.2 RESULTS AND DISCUSSION

The lipid binding properties of peptide 15 (SEQ ID NO:15) (which ispeptide 4 containing a Trp residue at position 10) are presented inTABLE VIII.

                  TABLE VIII                                                      ______________________________________                                        BINDING PROPERTIES OF PEPTIDE 15 (SEQ ID NO: 15)                                TO LIPIDIC VESICLES AS MEASURED BY FLUORESCENCE                               Lipid:Peptide                                                                 Molar Ratio (Ri) I/I.sub.o .sub.max (nm)                                    ______________________________________                                         0               0      332                                                      5 10.8 323                                                                    10 13.2 323.5                                                                 30 17.5 323                                                                  100 26.4 323                                                                  200 43.5 323                                                                ______________________________________                                    

The maximum of the tryptophane emission (λ_(max)) at 332 nm indicatesthat in the buffer at a concentration of 2 μM the peptide is slightlyself-associated. The peptide binds to the lipidic vesicles (EPC/Chol 5%)with a very high affinity as demonstrated by the burying of the Trp (themaximum of the Trp emission wavelength shifts from 332 nm down to 323nm) and the high fluorescence intensity exaltation (see TABLE VIII). Themaximum burying of the Tryptophane residue is obtained for a very lowlipid to peptide molar ratio lower than 5.

Other peptides which exhibited a high degree of helicity in the presenceof lipids (≧60% for unblocked peptides of ≧22 amino acids, or blockedpeptides of ≦18 amino acids; ≧40% for unblocked peptides of ≦18 aminoacids) as measured by circular dichroism as disclosed in Section 7.1,supra, also demonstrated good lipid binding. Of course, among all thepeptides selected by the circular dichroism screening, only the onesthat could be followed by fluorescence were tested for their lipidbinding properties.

7.3 NUCLEAR MAGNETIC RESONANCE (NMR)

This Example describes an NMR method for analyzing the structure of thecore peptides of the invention.

7.3.1 NMR SAMPLE PREPARATION

Samples were prepared by dissolving 5 mg of peptide in 90% H₂ O/10% D₂ Ocontaining trace amounts of 2,2-Dimethyl-2-sila-5-pentane sulfonate(DSS) as an internal chemical shift reference. Some of the samplescontained trifluoroethanol (TFE) (expressed as % vol). The total samplevolume was 500 μl and the concentration of peptide was approximately 5mM.

7.3.2 NMR SPECTROSCOPY

¹ H NMR spectra were acquired at 500 MHz using a Bruker DRX500spectrometer equipped with a B-VT2000 temperature control unit. One andtwo-dimensional experiments were recorded using standard pulsesequences. (Two Dimensional NMR Spectroscopy, Eds. W. R. Croasmun andRMK Carlson, 1994, VCH Publishers, New York, USA). Water suppression wasachieved with low power presaturation for 2 sec. Two-dimensionalexperiments were carried out in the phase sensitive mode using timeproportional phase incrementation (TPPI) and a spectral width of 6000 Hzin both dimensions. Typically, 40 scans were co-added for 400 t₁increments with 2048 data points. Data were processed using FELIX95software (Molecular Simulations) on an INDIGO2 workstation (SiliconGraphics). Data were zero-filled to give a 2K×2K data matrix andapodized by a 45° shifted squared sine-bell function.

7.3.3 NMR ASSIGNMENT

Complete proton resonance assignments were obtained by applying thesequential assignment technique using DQFCOSY, TOCSY and NOESY spectraas described in the literature (Wuthrich, NMR of Proteins and NucleicAcids, 1986, John Wiley & Sons, New York, USA). Secondary chemicalshifts were calculated for HN and Hα protons by subtracting thetabulated random coil chemical shifts (Wishart and Sykes, 1994, Method.Enz. 239:363-392) from the corresponding experimental values.

7.3.4 RESULTS AND DISCUSSION

General Consideration.

Amphipathic helical peptides tend to aggregate in aqueous solutions atthe high concentrations necessary for NMR spectroscopy, making itdifficult to obtain high resolution spectra. For example, NMR spectra ofexemplary core peptide 4 (SEQ ID NO:4) in water exhibit very broadlines. Thus, the resonances of each amino acid residue cannot beresolved. Addition of TFE to the sample improves the resolution of thespectra. TFE is known to solubilize peptides, and in addition stabilizeshelical conformations of peptides having helical propensity. Thefindings from NMR spectroscopy are demonstrated for peptide 4 (SEQ IDNO:4) as a representative example. The consensus 22-mer of Segrest (SEQID NO:75) was studied in comparison.

Secondary chemical shifts.

Proton chemical shifts of amino acids depend both on the type of residueand on the local secondary structure within a peptide or protein(Szlagyi, 1995, Progress in Nuclear Magnetic Resonance Spectroscopy27:325-443). Therefore, identification of regular secondary structure ispossible by comparing experimental shifts with tabulated values forrandom coil conformation.

Formation of an α-helix typically results in an up-field (negative)shift for the Hα resonance. Observation of an upfield Hα shift forseveral sequential residues is generally taken as evidence of a helicalstructure. The Hα secondary shifts for peptide 4 (SEQ ID NO:4) in 25%TFE at 295 K show a significant negative shift for residues 4 through 19(FIG. 9A), demonstrating a highly helical conformation. Smalldifferences are observed in the Hα chemical shifts of the consensus22-mer (SEQ ID NO:75) compared to peptide 4 (SEQ ID NO:4).

The chemical shifts of amide hydrogens of amino acid residues residingin regions of α-helix are also shifted upfield with respect to thechemical shifts observed for random coil. In addition, a periodicity ofthe HN shifts can be observed, and it reflects the period of the helicalturns. The amplitude of the shift variation along the sequence isrelated to the amphipathicity of a helical peptide. A higher hydrophobicmoment leads to a more pronounced oscillation (Zhou et al., 1992, J. Am.Chem. Soc. 114:4320-4326). The HN secondary shifts for peptide 4 (SEQ IDNO:4) in 25% TFE at 295 K show an oscillatory behavior in agreement withthe amphipathic nature of the helix (FIG. 9B). The amide chemical shiftpattern of the consensus 22-mer peptide (SEQ ID NO:75) differssignificantly from that of peptide 4 (SEQ ID NO:4). In particular,replacement of residues 5, 9 and 13 with Leu (L) results in chemicalshifts with a more pronounced periodicity in the case of peptide 4 (SEQID NO:4). The effect even extends to residue 17, and to a lesser extentto residue 21. From the NMR data, the existence of 5-6 turns in thesequence can be discerned. Thus, the replacement of three amino acidsaffects the folding of the peptide along the entire sequence. The NMRpattern clearly reflects the stronger amphipathic nature of peptide 4(SEQ ID NO:4) as compared to the consensus 22-mer peptide of Segrest(SEQ ID NO:75).

The secondary shift of an amide proton is influenced by the length ofthe hydrogen bond to the carbonyl oxygen one turn away from the helix.Therefore, the periodicity of observed chemical shift values reflectsdifferent hydrogen bond lengths. This difference is associated with anoverall curved helical shape of the helix backbone. The hydrophobicresidues are situated on the concave side. The secondary shifts ofpeptide 4 (SEQ ID NO:4) indicate a curved α-helical conformation.

7.3.5 PEPTIDES CONTAINING INTERNAL GLYCINES ARE HELICAL IN THE PRESENCEOF TFE

The three-dimensional structure of peptide 8 (SEQ ID NO:8) wasdetermined in the presence of TFE using Nuclear Overhauser Effects(NOEs) derived interproton distance constraints. In particular, thepresence of medium range NOE's (dαN₃, dαβ₃, daN₄) along the entiresequence are consistent with an overall α-helical structure. A family ofconformers was generated from the NMR data set and the structuressuperimpose well from residue 4 to 19 with a backbone RMSD<0.8 Å. Astereo ribbon representation of the average structure together with thebackbone trace of the 15 lowest energy conformers confirm a curvedhelical shape. In an approximate estimation, the bend results in a 20°angle between the N-terminal and the C-terminal half of the peptide. Thehydrophobic residues are mainly found on the concave side with theexception of Leu-5. A well defined hydrophobic cluster is centeredaround Phe-6 including Leu-3, Leu-9 and Leu-10. While the helical turnsstart around residue 3 at the N-terminus, the existence of many NOE's upto residue 22 indicate that the helix extends towards the end of theC-terminus.

8. EXAMPLE: LCAT ACTIVATION ASSAY

The peptides synthesized as described in Section 6, supra, were analyzedin vitro for their ability to activate LCAT. In the LCAT assay,substrate vesicles (small unilamellar vesicles or "SUVs") composed ofegg phophatidylcholine (EPC) or 1-Palmitoyl-2-oleyl-phosphatidylcholine(POPC) and radiolabelled cholesterol are preincubated with equivalentmasses either of peptide or ApoA-I (isolated from human plasma). Thereaction is initiated by addition of LCAT (purified from human plasma).Native ApoA-I, which was used as positive control, represents 100%activation activity. "Specific activity" (i.e., units of activity (LCATactivation)/unit of mass) of the peptides can be calculated as theconcentration of peptide that achieves maximum LCAT activation. Forexample, a series of concentrations of the peptide (e.g., a limitingdilution) can be assayed to determine the "specific activity" for thepeptide--the concentration which achieves maximal LCAT activation (i.e.,percentage conversion of cholesterol to cholesterol ester) at a specifictimepoint in the assay (e.g., 1 hr.). When plotting percentageconversion of cholesterol at, e.g., 1 hr., against the concentration ofpeptide used, the "specific activity" can be identified as theconcentration of peptide that achieves a plateau on the plotted curve.

8.1 PREPARATION OF SUBSTRATE VESICLES

The vesicles used in the LCAT assay are SUVs composed of eggphosphatidylcholine (EPC) or 1-palmitoyl-2-oleyl-phosphatidylcholine(POPC) and cholesterol with a molar ratio of 20:1. To prepare a vesiclestock solution sufficient for 40 assays, 7.7 mg EPC (or 7.6 mg POPC; 10μmol), 78 μg (0.2 μmol) 4-¹⁴ C-cholesterol, 116 μg cholesterol (0.3μmol) are dissolved in 5 ml xylene and lyophilized. Thereafter 4 ml ofassay buffer is added to the dry powder and sonicated under nitrogenatmosphere at 4° C. Sonication conditions: Branson 250 sonicator, 10 mmtip, 6×5 minutes; Assay buffer: 10 mM Tris, 0.14 M NaCl, 1 mM EDTA, pH7.4). The sonicated mixture is centrifuged 6 times for 5 minutes eachtime at 14,000 rpm (16,000×g) to remove titanium particles. Theresulting clear solution is used for the enzyme assay.

8.2 PURIFICATION OF LCAT

For the LCAT purification, dextran sulfate/Mg²⁺ treatment of humanplasma is used to obtain lipoprotein deficient serum (LPDS), which issequentially chromatographed on Phenylsepharose, Affigelblue,ConcanavalinA sepharose and anti-ApoA-I affinity chromatography, assummarized for a representative purification in TABLE IX, below:

                  TABLE IX                                                        ______________________________________                                        LCAT PURIFICATION                                                                        Total   Total Total                                                   Volume Protein Activity Yield Purification                                   Fraction (ml) (mg) (nmol CE/mg*hr) (%) (fold)                               ______________________________________                                        Plasma 550     44550   63706                                                    LPDS 500 31000 62620 98 1.4                                                   Phenyl 210 363 51909 82 100                                                   sepharose                                                                     Affigel 95 153 25092 39 115                                                   blue                                                                          ConA 43 36 11245 18 220                                                       sepharose                                                                     Anti-A-I 120 3.5 5500 9 1109                                                  Affinity                                                                    ______________________________________                                    

8.2.1 PREPARATION OF LPDS

To prepare LPDS, 500 ml plasma is added to 50 ml dextran sulfate(MW=500000) solution. Stir 20 minutes. Centrifuge for 30 minutes at 3000rpm (16,000×g) at 4° C. Use supernatant (LPDS) for further purification(ca. 500 ml).

8.2.2 PHENYLSEPHAROSE CHROMATOGRAPHY

The following materials and conditions were used for the phenylsepharosechromatography.

solid phase: Phenylsepharose fast flow, high subst. grade, Pharmacia

column: XK26/40, gel bed height: 33 cm, V=ca. 175 ml

flow rates: 200 ml/hr (sample)

wash: 200 ml/hr (buffer)

elution: 80 ml/hr (distilled water)

buffer: 10 mM Tris, 140 mM NaCl, 1 mM EDTA pH7.4, 0.01% sodium azide.

Equilibrate the column in Tris-buffer, add 29 g NaCl to 500 ml LPDS andapply to the column. Wash with several volumes of Tris buffer until theabsorption at 280 nm wavelength is approximately at the baseline, thenstart the elution with distilled water. The fractions containing proteinare pooled (pool size: 180 ml) and used for Affigelblue chromatography.

8.2.3 AFFIGELBLUE CHROMATOGRAPHY

The Phenylsepharose pool is dialyzed overnight at 4° C. against 20 mMTris-HCl, pH7.4, 0.01% sodium azide. The pool volume is reduced byultrafiltration (Amicon YM30) to 50-60 ml and loaded on an Affigelbluecolumn.

solid phase: Affigelblue, Biorad, 153-7301 column, XK26/20, gel bedheight: ca. 13 cm; column volume: approx. 70 ml.

flow rates: loading:

15 ml/h

wash: 50 ml/h

Equilibrate column in Tris-buffer. Apply Phenylsepharose pool to column.Start in parallel to collect fractions. Wash with Tris-buffer. Thepooled fractions (170 ml) were used for ConA chromatography.

8.2.4 ConA CHROMATOGRAPHY

The Affigelblue pool was reduced via Amicon (YM30) to 30-40 ml anddialyzed against ConA starting buffer (1 mM Tris HCl pH7.4; 1 mM MgCl₂,1 mM MnCl₂, 1 mM CaCl₂, 0.01% sodium azide) overnight at 4° C.

solid phase: ConA sepharose (Pharmacia)

column: XK26/20, gel bed height: 14 cm (75 ml)

flow rates:

loading 40 ml/h

washing (with starting buffer): 90 ml/h

elution: 50 ml/h, 0.2M 3949 Methyl-α-D-mannoside in 1 mM Tris, pH 7.4.

The protein fractions of the mannoside elutions were collected (110 ml),and the volume was reduced by ultrafiltration (YM30) to 44 ml. The ConApool was divided in 2 ml aliquots, which are stored at -20° C.

8.2.5 ANTI-ApoA-I AFFINITY CHROMATOGRAPHY

Anti-ApoA-I affinity chromatography was performed on Affigel-Hz material(Biorad), to which the anti-ApoA-I abs have been coupled covalently.

column: XK16/20, V=16 ml. The column was equilibrated with PBS pH 7.4.Two ml of the ConA pool was dialyzed for 2 hours against PBS beforeloading onto the column.

flow rates: loading: 15 ml/hour washing (PBS) 40 ml/hour.

The pooled protein fractions (V=14 ml) are used for LCAT assays.

The column is regenerated with 0.1 M. Citrate buffer (pH 4.5) to elutebound A-I (100 ml), and immediately after this procedure reequilibratedwith PBS.

8.3 RESULTS

The results of the LCAT activation assay are presented in TABLE X,infra.

                                      TABLE X                                     __________________________________________________________________________    LCAT ACTIVATION EXHIBITED BY EXEMPLARY CORE PEPTIDES                                                        ACTIVITY                                                                            He (%)                                                                            He (%)                                                                            He (%)                                                                            He (%)                          PEPTIDE  AMINO ACID SEQUENCE (%) LCAT free mics SUVs TFE                    __________________________________________________________________________    1    (SEQ ID NO: 1)                                                                         PVLDLFRELLNELLEZLKQKLK                                                                        120%  77  85  81  69                              2 (SEQ ID NO: 2) GVLDLFRELLNELLEALKQKLKK 105%                                 3 (SEQ ID NO: 3) PVLDLFRELLNELLEWLKQKLK 98% 70 95 80 95                       4 (SEQ ID NO: 4) PVLDLFRELLNELLEALKQKLK 93% 80 95 97 94                       5 (SEQ ID NO: 5) pVLDLFRELLNELLEALKQKLKK 90%                                  6 (SEQ ID NO: 6) PVLDLFRELLNEXLEALKQKLK 80% 57 93 70 99                       7 (SEQ ID NO: 7) PVLDLFKELLNELLEALKQKLK 83% 77 89 85 73                       8 (SEQ ID NO: 8) PVLDLFRELLNEGLEALKQKLK 83% 20 90 61 93                       9 (SEQ ID NO: 9) PVLDLFRELGNELLEALKQKLK 83%                                   10 (SEQ ID NO: 10) PVLDLFRELLNELLEAZKQKLK 79% 60 87 70 71                     11 (SEQ ID NO: 11) PVLDLFKELLQELLEALKQKLK 72%                                 12 (SEQ ID NO: 12) PVLDLFRELLNELLEAGKQKLK 70%                                 13 (SEQ ID NO: 13) GVLDLFRELLNEGLEALKQKLK 67%                                 14 (SEQ ID NO: 14) PVLDLFRELLNELLEALOQOLO 61% 70 96 80                        15 (SEQ ID NO: 15) PVLDLFRELWNELLEALKQKLK 60% 55 60 64 68                     16 (SEQ ID NO: 16) PVLDLLRELLNELLEALKQKLK 59%                                 17 (SEQ ID NO: 17) PVLELFKELLQELLEALKQKLK 59%                                 18 (SEQ ID NO: 18) GVLDLFRELLNELLEALKQKLK 58%                                 19 (SEQ ID NO: 19) pVLDLFRELLNEGLEALKQKLK 58%                                 20 (SEQ ID NO: 20) PVLDLFREGLNELLEALKQKLK 57%                                 21 (SEQ ID NO: 21) pVLDLFRELLNELLEALKQKLK 57%                                 22 (SEQ ID NO: 22) PVLDLFRELLNELLEGLKQKLK 54%                                 23 (SEQ ID NO: 23) PLLELFKELLQELLEALKQKLK 54%                                 24 (SEQ ID NO: 24) PVLDLFRELLNELLEALQKKLK 53%                                 25 (SEQ ID NO: 25) PVLDFFRELLNEXLEALKQKLK 51% 46 82  93                       26 (SEQ ID NO: 26) PVLDLFRELLNELLELLKQKLK 47%                                 27 (SEQ ID NO: 27) PVLDLFRELLNELZEALKQKLK 44% 72 92 82 81                     28 (SEQ ID NO: 28) PVLDLFRELLNELWEALKQKLK 40% 82 98 90 81                     29 (SEQ ID NO: 29) AVLDLFRELLNELLEALKQKLK 39%                                 30 (SEQ ID NO: 30) PVLDLFRELLNELLEALKQKLK.sup.t 38% 85 90 98 90                                                              31 (SEQ ID NO: 31)                                                           PVLDLFLELLNEXLEALKQKLK                                                        34% 49 98  90                   32 (SEQ ID NO: 32) XVLDLFRELLNELLEALKQKLK 33%                                 33 (SEQ ID NO: 33) PVLDLFREKLNELLEALKQKLK 33%                                 34 (SEQ ID NO: 34) PVLDZFRELLNELLEALKQKLK 32% 58 67 68 62                     35 (SEQ ID NO: 35) PVLDWFRELLNELLEALKQKLK 31% 49 59 61                            (sp)                                                                      36 (SEQ ID NO: 36) PLLELLKELLQELLEALKQKLK 31% 95 100   95                     37 (SEQ ID NO: 37) PVLDLFREWLNELLEALKQKLK 29% 65 75 76 73                     38 (SEQ ID NO: 38) PVLDLFRELLNEXLEAWKQKLK 29% 25 49 21 49                     39 (SEQ ID NO: 39) PVLDLFRELLEELLKALKKKLK 25% 66 69 68 72                     40 (SEQ ID NO: 40) PVLDLFNELLRELLEALQKKLK 25% 66 84 79 77                     41 (SEQ ID NO: 41) PVLDLWRELLNEXLEALKQKLK 25% 53 73 85 69                     42 (SEQ ID NO: 42) PVLDEFREKLNEXWEALKQKLK 25% 15 74 27 76                     43 (SEQ ID NO: 43) PVLDEFREKLWEXLEALKQKLK 25%                                 44 (SEQ ID NO: 44) pvldefreklneXlealkqklk 25% 20 86                           45 (SEQ ID NO: 45) PVLDEFREKLNEXLEALKQKLK 24% 24 84 25 86                     46 (SEQ ID NO: 46) PVLDLFREKLNEXLEALKQKLK 23% 30 86 58 85                     47 (SEQ ID NO: 47) ˜VLDLFRELLNEGLEALKQKLK 23%                           48 (SEQ ID NO: 48) pvLDLFRELLNELLEALKQKLK 22%                                 49 (SEQ ID NO: 49) PVLDLFRNLLEKLLEALEQKLK 22% 57 65 52 57                     50 (SEQ ID NO: 50) PVLDLFRELLWEXLEALKQKLK 21% 68 84 89 76                     51 (SEQ ID NO: 51) PVLDLFWELLNEXLEALKQKLK 20% 63 82 81 73                     52 (SEQ ID NO: 52) PVWDEFREKLNEXLEALKQKLK 20% sp sp sp                        53 (SEQ ID NO: 53) VVLDLFRELLNELLEALKQKLK 19%                                 54 (SEQ ID NO: 54) PVLDLFRELLNEWLEALKQKLK 19% 76 71 84 78                     55 (SEQ ID NO: 55) P˜˜˜LFRELLNELLEALKQKLK 19% 38 72                                                       78 75                           56 (SEQ ID NO: 56) PVLDLFRELLNELLEALKQKKK 18%                                 57 (SEQ ID NO: 57) PVLDLFRNLLEELLKALEQKLK 18%                                 58 (SEQ ID NO: 58) PVLDEFREKLNEXLEALKQKL˜ 18%                           59 (SEQ ID NO: 59) LVLDLFRELLNELLEALKQKLK 17%                                 60 (SEQ ID NO: 60) PVLDLFRELLNELLEALKQ˜˜˜ 16% 39 83                                                       66 84                           61 (SEQ ID NO: 61) PVLDEFRWKLNEXLEALKQKLK 16%                                 62 (SEQ ID NO: 62) PVLDEWREKLNEXLEALKQKLK 16% 15 85 43                        63 (SEQ ID NO: 63) PVLDFFREKLNEXLEALKQKLK 16%                                 64 (SEQ ID NO: 64) PWLDEFREKLNEXLEALKQKLK 15%                                 65 (SEQ ID NO: 65) ˜VLDEFREKLNEXLEALKQKLK 15%                           66 (SEQ ID NO: 66) PVLDLFRNLLEELLEALQKKLK 15% 64 82 66 70                     67 (SEQ ID NO: 67) ˜VLDLFRELLNELLEALKQKLK 14% 81 90 84 94                                                              68 (SEQ ID NO: 68)                                                           PVLDEFRELLKEXLEALKQKLK                                                        14%                             69 (SEQ ID NO: 69) PVLDEFRKKLNEXLEALKQKLK 13%                                 70 (SEQ ID NO: 70) PVLDEFRELLYEXLEALKQKLK 12% 27 78 33 66                     71 (SEQ ID NO: 71) PVLDEFREKLNELXEALKQKLK 11%                                 72 (SEQ ID NO: 72) PVLDLFRELLNEXLWALKQKLK 11% sp sp sp                        73 (SEQ ID NO: 73) PVLDEFWEKLNEXLEALKQKLK 10%                                 74 (SEQ ID NO: 74) PVLDKFREKLNEXLEALKQKLK 10%                                 75.sup.1/ (SEQ ID NO: 75) PVLDEFREKLNEELEALKQKLK 10% 18 28 23 55                                                             76 (SEQ ID NO: 76)                                                           PVLDEFRELLFEXLEALKQKLK                                                        9% 41 88  66                    77 (SEQ ID NO: 77) PVLDEFREKLNKXLEALKQKLK  9%                                 78 (SEQ ID NO: 78) PVLDEFRDKLNEXLEALKQKLK                                     79 (SEQ ID NO: 79) PVLDEFRELLNELLEALKQKLK  9%                                 80 (SEQ ID NO: 80) PVLDLFERLLNELLEALQKKLK  9%                                 81 (SEQ ID NO: 81) PVLDEFREKLNWXLEALKQKLK                                     82 (SEQ ID NO: 82) ˜˜LDEFREKLNEXLEALKQKLK  8%                     83 (SEQ ID NO: 83) PVLDEFREKLNEXLEALWQKLK                                     84 (SEQ ID NO: 84) PVLDEFREKLNELLEALKQKLK  7%                                 85 (SEQ ID NO: 85) P˜LDLFRELLNELLEALKQKLK  7% 58 61 64 69                                                              86 (SEQ ID NO: 86)                                                           PVLELFERLLDELLNALQKKLK                                                        7%                              87 (SEQ ID NO: 87) pllelikellqellealkqklk  7% 100  100   100                  88 (SEQ ID NO: 88) PVLDKFRELLNEXLEALKQKLK  7%                                 89 (SEQ ID NO: 89) PVLDEFREKLNEXLWALKQKLK  6%                                 90 (SEQ ID NO: 90) ˜˜˜DEFREKLNEXLEALKQKLK  6%                                                              91 (SEQ ID NO: 91)                                                           PVLDEFRELLNEXLEALKQKLK                                                        6% 43 100   100                 92 (SEQ ID NO: 92) PVLDEFRELYNEXLEALKQKLK  5%                                 93 (SEQ ID NO: 93) PVLDEFREKLNEXLKALKQKLK  5%                                 94.sup.2/ (SEQ ID NO: 94) PVLDEFREKLNEALEALKQKLK  5% 18 70 27 63                                                             95 (SEQ ID NO: 95)                                                           PVLDLFRELLNLXLEALKQKLK                                                        5%  sp sp                       96 (SEQ ID NO: 96) pvldlfrellneXlealkqklk  5% 52 85 63 81                     97 (SEQ ID NO: 97) PVLDLFRELLNELLE˜˜˜˜˜.ab                                                    out.˜  4%                 98 (SEQ ID NO: 98) PVLDLFRELLNEELEALKQKLK  2%                                 99 (SEQ ID NO: 99) KLKQKLAELLENLLERFLDLVP  2% 72 88 80 80                     100 (SEQ ID NO: 100) pvldlfrellnellealkqklk  2% 83 92  98                     101 (SEQ ID NO: 101) PVLDLFRELLNWXLEALKQKLK  2%  sp sp                        102 (SEQ ID NO: 102) PVLDLFRELLNLXLEALKEKLK  2% sp                            103 (SEQ ID NO: 103) PVLDEFRELLNEELEALKQKLK  1%                               104 (SEQ ID NO: 104) P˜˜˜˜˜˜˜L                                                    LNELLEALKQKLK  1% 21 49                                                       29 55                           105 (SEQ ID NO: 105) PAADAFREAANEAAEAAKQKAK  1% 29 28 32 65                   106 (SEQ ID NO: 106) PVLDLFREKLNEELEALKQKLK  0%                               107 (SEQ ID NO: 107) klkqklaellenllerfldlvp  0% sp sp  77                     108 (SEQ ID NO: 108) PVLDLFRWLLNEXLEALKQKLK  0% 28 55  54                     109.sup.3/ (SEQ ID NO: 109) PVLDEFREKLNERLEALKQKLK  0% 19 45 23 58                                                           110 (SEQ ID NO: 110)                                                         PVLDEFREKLNEXXEALKQKLK                                                        0%                              111 (SEQ ID NO: 111) PVLDEFREKLWEXWEALKQKLK  0%                               112 (SEQ ID NO: 112) PVLDEFREKLNEXSEALKQKLK  0%                               113 (SEQ ID NO: 113) PVLDEFREKLNEPLEALKQKLK  0%  6 22                         114 (SEQ ID NO: 114) PVLDEFREKLNEXMEALKQKLK  0%                               115 (SEQ ID NO: 115) PKLDEFREKLNEXLEALKQKLK  0%                               116 (SEQ ID NO: 116) PHLDEFREKLNEXLEALKQKLK  0%                               117 (SEQ ID NO: 117) PELDEFREKLNEXLEALKQKLK  0%                               118 (SEQ ID NO: 118) PVLDEFREKLNEXLEALEQKLK  0%                               119 (SEQ ID NO: 119) PVLDEFREKLNEELEAXKQKLK  0%                               120 (SEQ ID NO: 120) PVLDEFREKLNEELEXLKQKLK  0%                               121 (SEQ ID NO: 121) PVLDEFREKLNEELEALWQKLK  0%                               122 (SEQ ID NO: 122) PVLDEFREKLNEELEWLKQKLK  0%                               123 (SEQ ID NO: 123) QVLDLFRELLNELLEALKQKLK                                   124 (SEQ ID NO: 124) PVLDLFOELLNELLEALOQOLO                                   125 (SEQ ID NO: 125) NVLDLFRELLNELLEALKQKLK                                   126 (SEQ ID NO: 126) PVLDLFRELLNELGEALKQKLK                                   127 (SEQ ID NO: 127) PVLDLFRELLNELLELLKQKLK 47%                               128 (SEQ ID NO: 128) PVLDLFRELLNELLEFLKQKLK                                   129 (SEQ ID NO: 129) PVLELFNDLLRELLEALQKKLK                                   130 (SEQ ID NO: 130) PVLELFNDLLRELLEALKQKLK                                   131 (SEQ ID NO: 131) PVLELFKELLNELLDALRQKLK                                   132 (SEQ ID NO: 132) PVLDLFRELLENLLEALQKKLK                                   133 (SEQ ID NO: 133) PVLELFERLLEDLLQALNKKLK                                   134 (SEQ ID NO: 134) PVLELFERLLEDLLKALNQKLK                                   135 (SEQ ID NO: 135) DVLDLFRELLNELLEALKQKLK                                   136 (SEQ ID NO: 136) PALELFKDLLQELLEALKQKLK                                   137 (SEQ ID NO: 137) PVLDLFRELLNEGLEAZKQKLK                                   138 (SEQ ID NO: 138) PVLDLFRELLNEGLEWLKQKLK                                   139 (SEQ ID NO: 139) PVLDLFRELWNEGLEALKQKLK                                   140 (SEQ ID NO: 140) PVLDLFRELLNEGLEALOQOLO                                   141 (SEQ ID NO: 141) PVLDFFRELLNEGLEALKQKLK                                   142 (SEQ ID NO: 142) PVLELFRELLNEGLEALKQKLK                                   143 (SEQ ID NO: 143) PVLDLFRELLNEGLEALKQKLK*                                  144 (SEQ ID NO: 144) pVLELFENLLERLLDALQKKLK 111%  89 88  95                   145 (SEQ ID NO: 145) GVLELFENLLERLLDALQKKLK 100%  55 51  58                   146 (SEQ ID NO: 146) PVLELFENLLERLLDALQKKLK 86% 97 100  100  95                                                              147 (SEQ ID NO: 147)                                                         PVLELFENLLERLFDALQKKLK                                                        76%                             148 (SEQ ID NO: 148) PVLELFENLLERLGDALQKKLK 75% 10 76 23 80                   149 (SEQ ID NO: 149) PVLELFENLWERLLDALQKKLK 63% 28 54  47                     150 (SEQ ID NO: 150) PLLELFENLLERLLDALQKKLK 57%                               151 (SEQ ID NO: 151) PVLELFENLGERLLDALQKKLK 55%                               152 (SEQ ID NO: 152) PVFELFENLLERLLDALQKKLK 50%                               153 (SEQ ID NO: 153) AVLELFENLLERLLDALQKKLK 49%                               154 (SEQ ID NO: 154) PVLELFENLLERGLDALQKKLK 39% 13 76 25 80                   155 (SEQ ID NO: 155) PVLELFLNLWERLLDALQKKLK 38%                               156 (SEQ ID NO: 156) PVLELFLNLLERLLDALQKKLK 35%                               157 (SEQ ID NO: 157) PVLEFFENLLERLLDALQKKLK 30%                               158 (SEQ ID NO: 158) PVLELFLNLLERLLDWLQKKLK 30%                               159 (SEQ ID NO: 159) PVLDLFENLLERLLDALQKKLK 28%                               160 (SEQ ID NO: 160) PVLELFENLLERLLDWLQKKLK 28% 65 73 75 61                   161 (SEQ ID NO: 161) PVLELFENLLERLLEALQKKLK 27%                               162 (SEQ ID NO: 162) PVLELFENWLERLLDALQKKLK 27% 68 83 81                      163 (SEQ ID NO: 163) PVLELFENLLERLWDALQKKLK 26% 27 53  55                     164 (SEQ ID NO: 164) PVLELFENLLERLLDAWQKKLK 24% 37 66 51 61                   165 (SEQ ID NO: 165) PVLELFENLLERLLDLLQKKLK 23%                               166 (SEQ ID NO: 166) PVLELFLNLLEKLLDALQKKLK 22%                               167 (SEQ ID NO: 167) PVLELFENGLERLLDALQKKLK 18%                               168 (SEQ ID NO: 168) PVLELFEQLLEKLLDALQKKLK 17%                               169 (SEQ ID NO: 169) PVLELFENLLEKLLDALQKKLK 17%                               170 (SEQ ID NO: 170) PVLELFENLLEOLLDALQOOLO 17%                               171 (SEQ ID NO: 171) PVLELFENLLEKLLDLLQKKLK 16%                               172 (SEQ ID NO: 172) PVLELFLNLLERLGDALQKKLK 16%                               173 (SEQ ID NO: 173) PVLDLFDNLLDRLLDLLNKKLK 15%                               174 (SEQ ID NO: 174) pvlelfenllerlldalqkklk 13%                               175 (SEQ ID NO: 175) PVLELFENLLERLLELLNKKLK 13%                               176 (SEQ ID NO: 176) PVLELWENLLERLLDALQKKLK 11%                               177 (SEQ ID NO: 177) GVLELFLNLLERLLDALQKKLK 10%                               178 (SEQ ID NO: 178) PVLELFDNLLEKLLEALQKKLR  9%                               179 (SEQ ID NO: 179) PVLELFDNLLERLLDALQKKLK  8%                               180 (SEQ ID NO: 180) PVLELFDNLLDKLLDALQKKLR  8%                               181 (SEQ ID NO: 181) PVLELFENLLERWLDALQKKLK  8%                               182 (SEQ ID NO: 182) PVLELFENLLEKLLEALQKKLK  7%                               183 (SEQ ID NO: 183) PLLELFENLLEKLLDALQKKLK  6%                               184 (SEQ ID NO: 184) PVLELFLNLLERLLDAWQKKLK  4%                               185 (SEQ ID NO: 185) PVLELFENLLERLLDALQOOLO  3%                               186 (SEQ ID NO: 186) PVLELFEQLLERLLDALQKKLK                                   187 (SEQ ID NO: 187) PVLELFENLLERLLDALNKKLK                                   18B (SEQ ID NO: 188) PVLELFENLLDRLLDALQKKLK                                   189 (SEQ ID NO: 189) DVLELFENLLERLLDALQKKLK                                   190 (SEQ ID NO: 190) PVLEFWDNLLDKLLDALQKKLR                                   191 (SEQ ID NO: 191) PVLDLLRELLEELKQKLK* 100%                                 192 (SEQ ID NO: 192) PVLDLFKELLEELKQKLK* 100%  36   56                        193 (SEQ ID NO: 193) PVLDLFRELLEELKQKLK* 96% 34 88 87 87                      194 (SEQ ID NO: 194) PVLELFRELLEELKQKLK* 88% 38 93  93                        195 (SEQ ID NO: 195) PVLELFKELLEELKQKLK* 87%                                  196 (SEQ ID NO: 196) PVLDLFRELLEELKNKLK* 81%                                  197 (SEQ ID NO: 197) PLLDLFRELLEELKQKLK* 81% 43 70  69                        198 (SEQ ID NO: 198) GVLDLFRELLEELKQKLK* 80%                                  199 (SEQ ID NO: 199) PVLDLFRELWEELKQKLK* 76% 35 77 80 79                      200 (SEQ ID NO: 200) NVLDLFRELLEELKQKLK* 75%                                  201 (SEQ ID NO: 201) PLLDLFKELLEELKQKLK* 74%                                  202 (SEQ ID NO: 202) PALELFKDLLEELRQKLR* 70%                                  203 (SEQ ID NO: 203) AVLDLFRELLEELKQKLK* 66%                                  204 (SEQ ID NO: 204) PVLDFFRELLEELKQKLK* 63%                                  205 (SEQ ID NO: 205) PVLDLFREWLEELKQKLK* 60%                                  206 (SEQ ID NO: 206) PLLELLKELLEELKQKLK* 57%                                  207 (SEQ ID NO: 207) PVLELLKELLEELKQKLK* 50%                                  208 (SEQ ID NO: 208) PALELFKDLLEELRQRLK* 48%                                  209 (SEQ ID NO: 209) PVLDLFRELLNELLQKLK 47% 54 71 67 62                       210 (SEQ ID NO: 210) PVLDLFRELLEELKQKLK 46% 20 63 37 53                       211 (SEQ ID NO: 211) PVLDLFRELLEELOQOLO* 45%                                  212 (SEQ ID NO: 212) PVLDLFOELLEELOQOLK* 43%                                  213 (SEQ ID NO: 213) PALELFKDLLEEFRQRLK* 42%                                  214 (SEQ ID NO: 214) PVLDLFRELLEELKQKLK* 39%                                  215 (SEQ ID NO: 215) PVLDLFRELLEEWKQKLK* 38% 28 63 53 68                      216 (SEQ ID NO: 216) PVLELFKELLEELKQKLK 35%                                   217 (SEQ ID NO: 217) PVLDLFRELLELLKQKLK 30% 52 78 76 70                       218 (SEQ ID NO: 218) PVLDLFRELLNELLQKLK* 29%                                  219 (SEQ ID NO: 219) PVLDLFRELLNELWQKLK 24%                                   220 (SEQ ID NO: 220) PVLDLFRELLEELQKKLK 22% 27 64 54 64                       221 (SEQ ID NO: 221) DVLDLFRELLEELKQKLK* 12%                                  222 (SEQ ID NO: 222) PVLDAFRELLEALLQLKK  8%                                   223 (SEQ ID NO: 223) PVLDAFRELLEALAQLKK  8% 21 56 23 51                       224 (SEQ ID NO: 224) PVLDLFREGWEELKQKLK  8%                                   225 (SEQ ID NO: 225) PVLDAFRELAEALAQLKK  1%                                   226 (SEQ ID NO: 226) PVLDAFRELGEALLQLKK  1%                                   227 (SEQ ID NO: 227) PVLDLFRELGEELKQKLK*  0%                                  228 (SEQ ID NO: 228) PVLDLFREGLEELKQKLK*  0%                                  229 (SEQ ID NO: 229) PVLDLFRELLEEGKQKLK*  0%                                  230 (SEQ ID NO: 230) PVLELFERLLEDLQKKLK                                       231 (SEQ ID NO: 231) PVLDLFRELLEKLEQKLK                                       232 (SEQ ID NO: 232) PLLELFKELLEELKQKLK*                                      237.sup.4/ (SEQ ID NO: 237) LDDLLQKWAEAFNQLLKK 11% 30 66 45 --                238.sup.5/ (SEQ ID NO: 238) EWLKAFYEKVLEKLKELF* 19% 49 72 60 58                                                              239.sup.6/ (SEQ ID NO:                                                       239) EWLEAFYKKVLEKLKELF*                                                      11% 44 49  sp                   240 (SEQ ID NO: 240) DWLKAFYDKVAEKLKEAF* 10% 16 68 59 57                      241 (SEQ ID NO: 241) DWFKAFYDKVFEKFKEFF  8%                                   242 (SEQ ID NO: 242) GIKKFLGSIWKFIKAFVG  7%                                   243 (SEQ ID NO: 243) DWFKAFYDKVAEKFKEAF  5% 10 64 50                          244.sup.8/ (SEQ ID NO: 244) DWLKAFYDKVAEKLKEAF  5%  9 40 13 48                245 (SEQ ID NO: 245) DWLKAFYDKVFEKFKEFF  4% 38 77 70 sp                       246.sup.9/ (SEQ ID NO: 246) EWLEAFYKKVLEKLKELF  4% 18 44 47                   247 (SEQ ID NO: 247) DWFKAFYDKFFEKFKEFF  3%                                   248.sup.10/ (SEQ ID NO: 248) EWLKAFYEKVLEKLKELF  3% 18 45 13                  249.sup.11/ (SEQ ID NO: 249) EWLKAEYEKVEEKLKELF*                              250.sup.12/ (SEQ ID NO: 250) EWLKAEYEKVLEKLKELF*                              251.sup.13/ (SEQ ID NO: 251) EWLKAFYKKVLEKLKELF*                              252 (SEQ ID NO: 252) PVLDLFRELLEQKLK*                                         253 (SEQ ID NO: 253) PVLDLFRELLEELKQK*                                        254 (SEQ ID NO: 254) PVLDLFRELLEKLKQK*                                        255 (SEQ ID NO: 255) PVLDLFRELLEKLQK*                                         256 (SEQ ID NO: 256) PVLDLFRELLEALKQK*                                        257 (SEQ ID NO: 257) PVLDLFENLLERLKQK*                                        258 (SEQ ID NO: 258) PVLDLFRELLNELKQK*                                      __________________________________________________________________________     .sup.1/ Segrest's Consensus 22mer peptide (Anantharamaiah et al., 1990,       Arteriosclerosis 10(1):95-105).                                               .sup.2/ [A.sup.13 Consensus 22mer peptide (Anantharamaiah et al., 1990,       Arteriosclerosis 10(1):95-105).                                               .sup.3/ [R.sup.13 Consensus 22mer peptide (Anantharamaiah et al., 1990,       Arteriosclerosis 10(1):95-105).                                               .sup.4/ ID3 peptide (Labeur et al., 1997, Arteriosclerosis, Thrombosis an     Vascular Biology 17(3):580-588).                                              .sup.5/ Ac18AMOD-C(O)NH.sub.2 peptide (Epand et al., 1987, J. Biol. Chem.     262(19):9389-9396)                                                            .sup.6/ Ac18AM4-C(O)NH.sub.2 peptide (Brasseur, 1993, Biochim. Biophys.       Acta 1170:1-7).                                                               .sup.7/ 18L peptide (Segrest et al., 1990, Proteins: Structure, Function      and Genetics 8:103-117).                                                      .sup.8/ 18A peptide (Anantharamaiah et al., 1985, J. Biol. Chem.              260(18):10248-10255).                                                         .sup.9/ 18AM4 peptide (Rosseneu et al., WO93/25581; Corijn et al., 1993,      Biochim. Biophys. Acta 1170:8-16).                                            .sup.10/ [Glu.sup.1,8 ; Leu.sup.5,11,7 ]18A peptide (Epand et al., 1987,      J. Biol. Chem. 262(19):9389-9396).                                            .sup.11/ Ac18AM3-C(O)NH.sub.2 (Rosseneu et al., WO93/25581).                  .sup.12/ Ac18AM2-C(O)NH.sub.2 (Rosseneu et al., WO93/25581).                  .sup.13/ Ac18AM1-C(O)NH.sub.2 (Rosseneu et al., WO93/25581).             

In TABLE X, * indicates peptides that are N-terminal acetylated andC-terminal amidated; .Arrow-up bold. indicates peptides that areN-terminal dansylated; sp indicates peptides that exhibited solubilityproblems under the experimental conditions; X is Aib; Z is Nal; O isOrn; He (%) designates percent helicity; mics designates micelles; and ˜indicates deleted amino acids.

9. EXAMPLE: PHARMCOKINETICS OF THE ApoA-I AGONISTS

The following experiments demonstrate that the ApoA-I agonists arestable in the circulation and associate with the HDL component ofplasma.

In particular, radioactively labeled peptide 4 injectedintraperitoneally into mice associated with the HDL component andremained stable for at least 6 hours. When added to human plasma (exvivo) peptide 4 also associated with the HDL component.

9.1. SYNTHESIS OF RADIOLABELED PEPTIDES

Radiolabeled peptides 4 (SEQ ID NO:4) and 8 (SEQ ID NO:8) weresynthesized by coupling ¹⁴ C-labeled Fmoc-Pro as the N-terminal aminoacid.

L-[U-¹⁴ C]Proline, specific activity 9.25 GBq/mmol, was used for thesynthesis of labeled Fmoc-L-Proline. The synthesis was carried outaccording to Lapatsanis, Synthesis, 1983, 671-173. Briefly, 250 μM (29.6mg) of unlabeled L-Proline was dissolved in 225 μl of a 9% Na₂ CO₃solution and added to a solution (9% Na₂ CO₃) of 9.25 MBq (250 μM) ¹⁴ Clabeled L-Proline. The liquid was cooled down to 0° C., mixed with 600μM (202 mg) 9-fluorenylmethyl-N-succinimidylcarbonate (Fmoc-OSu) in 0.75ml DMF and shaken at room temperature for 4 hr. Thereafter the mixturewas extracted with Diethylether (2×5ml) and chloroform (1×5 ml), theremaining aqueous phase was acidified with 30% HCl and extracted withchloroform (5×8 ml). The organic phase was dried over Na₂ SO₄, filteredoff and the volume was reduced under nitrogen flow to 5 ml. The puritywas estimated by TLC (CHCl₃ :MeOH:Hac, 9:1:0.1 v/v/v, stationary phaseHPTLC silicagel 60, Merck, Germany) and showed a single peak (UVdetection, radiochemical purity:Linear Analyzer, Berthold, Germany);reaction yield: 90% (determined by LSC).

The chloroform solution containing ¹⁴ C-Fmoc Proline was used directlyfor peptide synthesis. A peptide resin containing amino acids 2-22,synthesized automatically as described in Section 6, was used for thesynthesis. The sequence of the peptide was determined by Edmandegradation. The coupling was performed as described in Section 6.1(manual coupling of Fmoc-Nal), except that HATU(O-(7-azabenzotriazol-1-yl)-1-,1,3,3-tetramethyluroniumhexafluorophosphate)was used instead of TBTU. A second coupling with unlabeled Fmoc-L-Prowas carried out manually as described in Section 6.1. Peptide cleavageand deprotection were as described in Section 6.4. The specificactivities of labeled peptides were as follows: ##EQU3##

9.2. PHARMACOKINETICS IN MICE

In each experiment, 2.5 mg/kg radiolabeled peptide was injectedintraperitoneally into mice which were fed normal mouse chow or theatherogenic Thomas-Harcroft modified diet (resulting in severelyelevated VLDL and IDL cholesterol). Blood samples were taken at multipletime intervals for assessment of radioactivity in plasma. Results aresummarized in TABLE XI, below.

                  TABLE XI                                                        ______________________________________                                        HALF-LIFE OF PEPTIDE 4 (SEQ ID NO: 4) IN MICE                                                               HALF-LIFE                                         SPECIES PREPARATION (hours)                                                 ______________________________________                                        C57BL/6       peptide 4/PBS                                                                             5.72.sup.1                                            (normal diet)                                                                 C57B1/6 peptide 4/PBS 5.98.sup.2                                              (high fat diet) peptide 4/POPC 6.39.sup.4                                      complex.sup.3                                                                ApoE.sup.6 peptide 4/PBS 14.2.sup.5                                         ______________________________________                                         .sup.1 The maximum serum concentration reached was 25.7% injected cpm at      1.9 hours following injection. r.sup.2 = 0.960.                               .sup.2 The maximum serum concentration reached was 20.3% injected cpm at      2.98 hours following injection. r.sup.2 = 0.973.                              .sup.3 Complexes consisting of phospholipid (POPC) and peptide 4 were         prepared using the cholate dialysis method.                                   .sup.4 The maximum serum concentration reached was 39.4% injected cpm at      4.2 hours following injection. r.sup.2 = 0.893.                               .sup.5 The maximum serum concentration reached was 17.4% injected cpm at      1.46 hours following injection. r.sup.2 = 0.973.                              .sup.6 ApoE Knockout.                                                    

9.3. STABILITY IN HUMAN SERUM

9.3.1. EXPERIMENTAL METHODS

100 μg of ¹⁴ C-labeled peptide 4 (SEQ ID NO:4) prepared as described inSection 9.1, supra, was mixed with 2 mL of fresh human plasma (at 37°C.) and delipidated either immediately (control sample) or after 8 daysof incubation at 37° C. (test sample). Delipidation was carried out byextracting the lipids with an equal volume of 2:1 (v/v)chloroform:methanol.

The samples were loaded onto a reverse-phase C₁₈ HPLC column and elutedwith a linear gradient (25-58% over 33 min.) of acetonitrile (containing0.1% TFA). Elution profiles were followed by absorbance (220 nm) andradioactivity.

9.3.2. RESULTS

The control sample eluted as a single peak with a retention time ofapproximately 30 min. All of the radioactivity eluted in this peak.

The test sample eluted as two peaks: one having a retention time ofabout 3 min., the other having a retention time of about 30 min. The 3min. peak (degraded peptide) accounted for approximately 15% of thetotal radioactivity loaded onto the column. The remainder of theradioactivity eluted with the 30 min. peak (intact peptide), indicatingthat peptide 4 (SEQ ID NO:4) is extremely stable to human serum--morethan 80% of the peptide 4 remained intact even after an 8 day incubationin human serum.

9.4. FORMATION OF PRE-β LIKE PARTICLES

9.4.1. EXPERIMENTAL METHOD

Human HDL was isolated by KBr density ultra centrifugation at densityd=1.21 g/ml to obtain top fraction followed by Superose 6 gel filtrationchromatography to separate HDL from other lipoproteins. Isolated HDL wasadjusted to a final concentration of 1.0 mg/ml with physiological salinebased on protein content determined by Bradford protein assay. Analiquot of 300 μl was removed from the isolated HDL preparation andincubated with 100 μl ¹⁴ C-labeled peptide 4 (0.2-1.0 μg/μl) for twohours at 37° C. Five separate incubations were analyzed including ablank containing 100 μl physiological saline and four dilutions of ¹⁴C-labeled peptide 4: (i) 0.20 μg/μl peptide:HDL ratio=1:15; (ii) 0.30μg/μl peptide:HDL ratio=1:10; (iii) 0.60 μg/μl peptide:HDL ratio=1:5;and (iv) 1.00 μg/μl peptide:HDL ratio=1:3. Following the two hourincubation, a 200 μl aliquot of the sample (total volume=400 μl) wasloaded onto a Superose 6 gel filtration column for lipoproteinseparation and analysis, and 100 μl was used to determine totalradioactivity loaded. The conditions for all FPLC chromatographies wereas described in Section 9.4, infra.

9.4.2. RESULTS

The results are presented in TABLE XII, infra, and FIGS. 9A-9F.

                                      TABLE XII                                   __________________________________________________________________________    DISPLACEMENT OF HUMAN APOA-I FROM ISOLATED                                      HUMAN HDL BY .sup.14 C-LABELED PEPTIDE 4 (SEQ ID NO:4)                                              .sup.14 C                                                                         .sup.14 C                                           Peak #1  loaded column Recovery                                                      1a  1b Peak #2                                                                           Peak #3                                                                           (cpm)                                                                             (cpm)                                                                             (%)                                           __________________________________________________________________________    P:LP = 1:15                                                                         EV 30.5                                                                   control AUC 9.95                                                              peptide EV 29.4 30.9                                                           AUC 4.09 6.26                                                                 .sup.14 C 7,920   1,622 10,361 9,542 92.1                                    P:LP = 1:10 EV 30.7                                                           control AUC 9.15                                                              peptide EV 30.7  33.0                                                          AUC 8.42  0.67                                                                .sup.14 C N/A   N/A N/A N/A N/A                                              P:LP = 1:5 EV 30.9                                                            control AUC 9.87                                                              peptide EV 30.8  34.3                                                          AUC 8.19  1.53                                                                .sup.14 C 20,768   5,281 28,794 26,049 90.5                                  P:LP = 1:3 EV 30.5                                                            control AUC 10.22                                                             peptide EV 30.6  34.3                                                          AUC 7.93  1.97                                                                .sup.14 C 43,495   7,832 54,593 51,321 94                                  __________________________________________________________________________     P:LP = Peptide to lipoprotein ratio based on mass (μg)                     EV = Elution volume (ml)                                                      AUC = Area under the curve (Peakfit program)                                  .sup.14 C = Cpms determined from radiometric counting                         Peak #1 = Fractions 29 to 33                                                  Peak #2 = Fractions 34 to 35                                                  Peak #3 = Fractions 41 to 47                                             

From the data presented in TABLE XII, it is evident that greater than90% of the radioactivity was recovered from the column after separationof the lipoprotein particles. At low concentration of peptide (i.e.,peptide:HDL mass ratio of 1:15), the HDL peak was split into twoseparate peaks (FIG. 9A), suggesting that an interaction between thepeptide and HDL occurred which resulted in some remodeling of thelipoprotein particle, but since no displaced peak was noted theinteraction was not sufficient to displace native ApoA-I. As theconcentration of peptide was increased, a displacement of ApoA-I wasobserved which increased with increasing concentration of peptide (FIGS.9B-9D). In addition, all chromatographic runs showed a third peakdetected by radiometric counting (except the one with a mass ratio of1:10, for which results are not shown) which is consistent with theelution volume of free peptide (FIG. 9E).

To further analyze the effect of peptide concentration on theinteraction of ¹⁴ C-labeled peptide 4 and HDL, a difference plot wasgenerated from the four chromatographic runs (FIGS. 9A-9D) and is shownin FIG. 9F. The difference plot demonstrates the shift in the HDL peakas well as the increased displacement of ApoA-I with increasing peptideconcentration. The displacement of ApoA-I results in the formation ofpre-β like particles.

9.5. ASSOCIATION OF PEPTIDE 4 WITH HUMAN LIPOPROTEINS 9.5.1.EXPERIMENTAL METHODS

The association of peptide 4 (SEQ ID NO:4) with human lipoproteinfractions was determined by incubating ¹⁴ C-labeled peptide 4 (SEQ IDNO:4) with each lipoprotein class (HDL, LDL and VLDL) and a mixture ofthe different lipoprotein classes.

HDL, LDL and VLDL were isolated by KBr density gradientultracentrifugation at d=1.21 g/ml and purified by FPLC on a Superose 6Bcolumn size exclusion column (chromatography was carried out with a flowrate of 0.7 ml/min. and a running buffer of 10 mM Tris (pH 8), 115 mMNaCl, 2 mM EDTA and 0.01% NaN₃). ¹⁴ C-labeled peptide 4 was incubatedwith HDL, LDL and VLDL at a peptide:phospholipid ratio of 1:5 (massratio) for 2 h at 37° C. The required amount of lipoprotein (volumesbased on amount needed to yield 1000 μg) was mixed with 0.2 ml ofpeptide stock solution (1 mg/ml) and the solution was brought up to 2.2ml using 0.9% of NaCl according to TABLE XIII:

                  TABLE XIII                                                      ______________________________________                                        PREPARATION OF PEPTIDE-LIPOPROTEIN SAMPLES                                                                         .sup.14 C-                                     labeled                                                                   HDL LDL VLDL 0.9% NaCl peptide 4 Total                                        (ml) (ml) (ml) (ml) (ml) (ml)                                               ______________________________________                                        Control                      2.0     0.2    2.2                                 LP/Peptide 0.4 0.3 0.9 0.4 0.2 2.2                                            HDL/Peptide 0.4   1.6 0.2 2.2                                                 LDL/Peptide  0.3  1.7 0.2 2.2                                                 VLDL/Peptide   0.9 1.1 0.2 2.2                                              ______________________________________                                    

After incubating for 2 hr. at 37° C., an aliquot (0.1 ml) was removedfor liquid scintillation counting to determine the total radioactivity,the density of the remaining incubation mixture was adjusted to 1.21g/ml with KBr, and the samples centrifuged at 100,000 rpm (300,000 g)for 24 hours at 4° C. in a TLA 100.3 rotor using a Beckman tabletopultracentrifuge. The resulting supernatant was fractionated by removing0.3 ml aliquots from the top of each sample for a total of 5 fractions,and 0.05 ml of each fraction was used for liquid scintillation counting.The top two fractions contain the floating lipoproteins, the otherfractions (3-5) correspond to proteins/peptides in solution.

9.5.2. RESULTS

The results are presented in TABLE XIV, infra. The incubation of ¹⁴C-labeled peptide 4 with the isolated lipoprotein fractions revealed astrong association with HDL (88% of total radioactivity in fractions 1and 2) as well as with VLDL (85%), and a substantially weaker affinityto LDL (66%). The incubation mixture consisting of all lipoproteinfractions and ¹⁴ C-labeled peptide 4 (LP/Peptide) showed 88% of thelabeled peptide associated with the lipoprotein fraction. FPLC analysisof the LP/peptide incubation mixture demonstrated that nearly all of the¹⁴ C-labeled peptide 4 was associated with the HDL fraction, indicatinga high selectivity for this lipoprotein class.

                  TABLE XIV                                                       ______________________________________                                        ASSOCIATION OF PEPTIDE 4 (SEQ ID NO: 4)                                         WITH VARIOUS LIPIDS                                                                              LP/    HDL/   LDL/   VLDL/                                 Fraction Control Peptide Peptide Peptide Peptide                            ______________________________________                                        1       650      60250    61510  43740  42920                                   2 1000 12990 17620 15140 19280                                                3 6590 3810 2260 11880 3930                                                   4 36550 1510 3130 10860 2600                                                  5 34380 5090 5450 7710 4550                                                   Total 79170 83650 89970 89330 73280                                           Total* 88550 96646 98494 100452 97306                                         Recovery 89 87 91 89 75                                                       (%)                                                                         ______________________________________                                         *before centrifugation                                                   

9.6. PEPTIDE 4 (SEQ ID NO:4) SELECTIVELY BINDS HDL LIPIDS IN HUMANPLASMA 9.6.1. EXPERIMENTAL METHOD

Human plasma (2 ml) was incubated with 20, 40, 60, 80, and 100 μg of ¹⁴C-labeled peptide peptide 4 for 2 hr. at 37° C. The lipoproteins wereseparated by adjusting the density to 1.21 g/ml and centrifugation inTLA 100.3 rotor at 100,000 rpm (300,000 g) for 36 hr. at 4° C. The top900 μl (in 300 μl fractions) was taken for the analysis. 50 μl from each300 μl fraction was counted for radioactivity and 200 μl from eachfraction was analyzed by FPLC (Superose 6/Superose 12 combinationcolumn).

9.6.2. RESULTS

The amount of radioactivity recovered for each fraction is presented inTABLE XV, infra. Most of the radioactivity was recovered in the topthree fractions. All the lipoprotein separation profiles of the plasmasample incubated with ¹⁴ C-labeled peptide 4 (SEQ ID NO:4) (not shown)indicate that, in every case, almost all of the radioactivity is presentin the HDL fraction. Thus, although other lipoproteins are present inthe human serum, peptide 4 (SEQ ID NO:4) exhibits highly selectivebinding to HDL.

                  TABLE XV                                                        ______________________________________                                        RADIOACTIVITY RECOVERED FROM INCUBATION OF                                      .sup.14 C-LABELED PEPTIDE 4 WITH HUMAN SERUM                                  Peptide    Fraction                                                                              Radioactivity (cpm*)                                                                        Recovery**                                 (μg)  No.     in 50 μl                                                                              in 300 μl                                                                         (%)                                        ______________________________________                                        20       1       1407       8442   71.6                                          2 405 2430 20.6                                                               3 152 912 7.7                                                                40 1 2538 15228 70.4                                                           2 721 4326 20                                                                 3 347 2082 9.6                                                               60 1 4492 26952 75.5                                                           2 1107 6642 18.6                                                              3 354 2124 5.9                                                               80 1 4715 28290 60                                                             2 2251 13506 28.6                                                             3 851 5346 11.3                                                              100 1 5424 32544 67.5                                                          2 1708 10248 21.2                                                             3 907 5442 11.3                                                            ______________________________________                                         *Value for 300 μl was obtained by multiplying the 50 μl value by 6.     **Based on the initial total counts used for mixing with plasma.         

10. EXAMPLE: THE ApoA-I AGONISTS PROMOTE CHOLESTEROL EFFLUX

HepG2 hepatoma cells were plated into 6-well culture dishes and grown toconfluence. Cells were labeled with ³ H-cholesterol by drying thecholesterol, then adding 1% bovine serum albumin (BSA) in phosphatebuffered saline (PBS), sonicating the solution, and adding 0.2 mllabeling solution and 1.8 ml growth medium to the cells, so that eachwell contained 2 μCi of radioactivity. Cells were incubated for 24 hwith the labeling medium.

Peptide (or protein):DMPC complexes were prepared at a 1:2 peptide (orprotein):DMPC ratio (w:w). To prepare the complexes, peptide 4 (SEQ IDNO:4) or native human ApoA-I protein was added to a DMPC solution in PBSand incubated at room temperature overnight, by which time the solutionhas clarified. Peptide or protein concentration in the final solutionwas 1 mg/ml.

Labeling media was removed from the cells and the cells were washed withPBS prior to addition of complexes. 1.6 ml of growth medium was added toeach well, followed by peptide (or protein):DMPC complex and sufficientPBS to bring the final volume to 2 ml per well. The final peptide orApoA-I concentrations were 1, 2.5, 5, 7.5 and 25 μg/ml medium. After 24hours of incubation at 37° C., the medium was removed, and the cellswashed with 2 ml of 1% BSA/PBS, followed by 2 washes with 2 ml each ofPBS. The amount of ³ H-cholesterol effluxed into the medium wasdetermined by liquid scintillation counting.

The results demonstrate that peptide 4 (SEQ ID NO:4) was more efficientat cholesterol efflux than ApoA-I.

11. EXAMPLE: USE OF THE ApoA-I AGONISTS IN ANIMAL MODEL SYSTEMS

The efficacy of the ApoA-I agonists of the invention was demonstrated inrabbits. The results show that administration of the ApoA-I agonistsincreases serum concentration of HDL-like particles.

11.1. PREPARATION OF THE PHOSPHOLIPID/PEPTIDE COMPLEXES

Small discoidal particles consisting of phospholipid (DPPC) and peptidewere prepared following the cholate dialysis method. The phospholipidwas dissolved in chloroform and dried under a stream of nitrogen. Thepeptide was dissolved in buffer (saline) at a concentration of 1-2mg/ml. The lipid film was redissolved in buffer containing cholate (43°C.) and the peptide solution was added at a 3:1 phospholipid/peptideweight ratio. The mixture was incubated overnight at 43° C. and thendialyzed at 43° C. (24 hr.), room temperature (24 hr.) and 4° C. (24hr.), with three changes of buffer (large volumes) at temperature point.The complexes were filter sterilized (0.22 μm) for injection and storageat 4° C.

11.2. ISOLATION AND CHARACTERIZATION OF THE PEPTIDE/PHOSPHOLIPIDPARTICLES

The particles were separated on a gel filtration column (Superose 6 HR).The position of the peak containing the particles was identified bymeasuring the phospholipid concentration in each fraction. From theelution volume, the Stokes radius was determined. The concentration ofpeptide in the complex was determined by measuring the phenylalaninecontent (by HPLC) following a 16 hr. acid hydrolysis.

11.3. INJECTION IN THE RABBIT

Male New Zealand White rabbits (2.5-3 kg) were injected intravenouslywith a dose of phospholipid/peptide complex (5 or 10 mg/kg bodyweight,expressed as peptide) in a single bolus injection not exceeding 10-15ml. The animals were slightly sedated before the manipulations. Bloodsamples (collected on EDTA) were taken before and 5, 15, 30, 60, 240 and1440 minutes after injection. The hematocrit (Hct) was determined foreach sample. Samples were aliquoted and stored at -20° C. beforeanalysis.

11.4. ANALYSIS OF THE RABBIT SERA

Plasma Lipids. The total plasma cholesterol, plasma triglycerides andplasma phospholipids were determined enzymatically using commerciallyavailable assays according to the manufacturer's protocols (BoehringerMannheim, Mannheim, Germany and Biomerieux, 69280, Marcy-L'etoile,France).

Lipoprotein Profiles. The plasma lipoprotein profiles of the fractionsobtained after the separation of the plasma into its lipoproteinfractions were determined by spinning in a sucrose density gradient. Thefractions were collected and in each individual fraction thephospholipid and cholesterol content was measured enzymatically.

11.5. RESULTS

The lipoprotein profile of rabbits injected with 10 mg/kg peptide 4 (SEQID NO:4) (in the form of peptide/DPPC complexes) as a function of timeis shown in FIG. 10. A substantial increase in cholesterol of the HDLcholesterol fractions (fractions >1.06 mg/ml) is apparent at 5 min.following injection and lasts for approximately 1 hr.

The cholesterol of the combined HDL fractions obtained by densitygradient ultracentrifugation is presented in TABLE XVI, below. Thehighest increase of HDL cholesterol (31.3%) occurred 15 min. afteradministration.

These data indicate that administration of peptide 4/DPPC complexes (10mg/kg) induces rapid and efficient mobilization of peripheralcholesterol.

                  TABLE XVI                                                       ______________________________________                                        HDL CHOLESTEROL IN RABBITS FOLLOWING                                            ADMINISTRATION OF 10 mg/kg PEPTIDE 4 (SEQ ID NO:4)                                                      Increase in HDL                                     Time (min.) HDL Cholesterol Cholesterol (%)                                 ______________________________________                                        0           325                                                                  408 25.2                                                                     15 428 31.3                                                                   60 387 18.9                                                                   240 291 -10.7                                                                 1440 347 6.6                                                                ______________________________________                                    

The dose-dependency of the peptide 4/DPPC complexes is shown in TABLEXVII, below. Based on these two time points, an approximately lineardose-dependency was observed.

                  TABLE XVII                                                      ______________________________________                                        DOSE-DEPENDENCY OF HDL CHOLESTEROL LEVELS                                       FOLLOWING ADMINISTRATION OF PEPTIDE 4/DPPC                                    COMPLEXES                                                                                HDL Cholesterol                                                                           HDL Cholesterol                                                                         HDL Cholesterol                              Dose (5 min.) (15 min.) (60 min.)                                           ______________________________________                                        5 mg/kg  33.7        28          60                                             5 mg/kg 49 40 20                                                              Mean Increase 41.4 35.0 40.0                                                  (%)                                                                           10 mg/kg 60.7 75 121.3                                                        10 mg/kg 35 42.5 35.6                                                         Mean Increase 47.9 58.8 78.5                                                  (%)                                                                         ______________________________________                                    

The percent increase in HDL cholesterol following injection of 5 mg/kgpeptide 1 (SEQ ID NO:1) (in the form of peptide/DPPC complexes) or 10mg/kg peptide 3 (SEQ ID NO:3) (in the form of peptide/DPPC complexes) isshown in TABLE XVIII, below.

                  TABLE XVIII                                                     ______________________________________                                                       Increase (%)                                                                            Increase (%)                                            (Peptide 1, (Peptide 3,                                                      Time (min) 5 mg/kg) 10 mg/kg)                                               ______________________________________                                        15             24.3      84                                                     30 95 71                                                                      60 40.7 60                                                                    240 29 14.5                                                                 ______________________________________                                    

These experiments demonstrate the ability of the ApoA-I agonists of theinvention to increase HDL-cholesterol. A substantial increase in HDLcholesterol is observed even 4 hr. following administration for peptide1 (SEQ ID NO:1) and peptide 3 (SEQ ID NO:3).

12. EXAMPLE: PREPARATION OF PEPTIDE-LIPID COMPLEX BY CO-LYOPHILIZATIONAPPROACH

The following protocol was utilized to prepare peptide-lipid complexes.

One mg of peptide 4 (SEQ ID NO:4) peptide was dissolved in 250 μl HPLCgrade methanol (Perkin Elmer) in a one ml clear glass vial with cap(Waters #WAT025054). Dissolving of the peptide was aided by occasionalvortexing over a period of 10 minutes at room temperature. To thismixture an aliquot containing either 1, 2, 3, 4, 5, 7.5, 10 or 15 mgdipalmitoyl phosphatidylcholine (DPPC; Avanti Polar Lipids, 99% Purity,product #850355) from a 100 mg/ml stock solution in methanol was added.The volume of the mixture was brought to 400 μl by addition of methanol,and the mixture was further vortexed intermittently for a period of 10minutes at room temperature. To each tube 200 μl of xylene(Sigma-Aldrich 99% pure, HPLC-grade) was added and the tubes werevortexed for 10 seconds each. Two small holes were punched into the topsof each tube with a 20 gauge syringe needle, the tubes were frozen for15 seconds each in liquid nitrogen, and the tubes were lyophilizedovernight under vacuum. To each tube 200 ml of 0.9% NaCl solution wasadded. The tubes were vortexed for 20 seconds each. At this time thesolutions in the tubes were milky in appearance. The tubes were thenincubated in a water bath for 30 minutes at 41° C. The solutions in allof the tubes became clear (i.e., similar to water in appearance) exceptfor the tube containing 15 mg DPPC, which remained cloudy.

The following protocol was used to prepare a greater amount ofpeptide-lipid complexes for in vivo experiments.

Peptide 4 (SEQ ID NO:4) (22.4 mg) was dissolved in methanol at aconcentration of 3.5 mg/ml by incubation for several minutes and mixingby vortex intermittently. To this solution was added dipalmitoylphosphatidylcholine (DPPC) in methanol (100 mg/ml stock solution) suchthat the final ratio of DPPC/peptide was 2.5:1 (weight/weight). Thissolution was mixed by vortexing. Xylene was added to this solution to afinal concentration of 36%. Aliquots of the resulting solution wereremoved for later analysis by gel filtration chromatography. Thesolutions were frozen in liquid nitrogen and lyophilized to dryness byvacuum. An aliquot containing 20 mg peptide and 50 mg DPPC wasrehydrated in sterile saline solution (0.9% NaCl), mixed, and heated to41° C. for several minutes until a clear solution of reconstitutedpeptide phospholipid complexes resulted.

12.1. CHARACTERIZATION OF COMPLEXES BY SUPEROSE 6 GEL FILTRATIONCHROMATOGRAPHY

Peptide-phospholipid complexes containing ¹⁴ C-labeled peptide 4 (SEQ IDNO:4) (specific radioactivity 159,000 DPM/mg peptide by weight, assuming50% peptide content) were prepared by colyophilization as described inthe text. The preparation contained 1 mg peptide and 4 mg DPPC byweight. After reconstituting the complexes in 200 μl 0.9% NaCl, 20 μl(100 μg) of the complexes were applied to a Pharmacia Superose 6 columnusing 0.9% NaCl as the liquid phase at a flow rate of 0.5 ml/minute.After a 5 ml delay (column void volume=7.7 ml), 1 ml fractions werecollected. Aliquots containing 20 μl of the fractions were assayed forphospholipid content using the bioMerieux Phospholipides Enzymatique PAP150 kit (#61491) according to the instructions supplied by themanufacturer. The remainders of each fraction were counted for 3 minutesin a Wallach 1410 liquid scintillation counter (Pharmacia) using theEasy Count program. The vast majority of both phospholipid and peptidewere recovered together in a few fractions with peaks at approximately16 ml. The UV absorbance profile for this sample (not shown) indicatesthat the complexes elute from the column at a volume of 14.7 ml. Thediscrepancy between the elution volume as measured byradioactivity/phospholipid assay and UV absorbance is due to the 1.3 mldead volume of tubing between the UV absorbance flow cell and thefraction collector outlet. This elution volume corresponds to a Stokes'diameters of 114 Angstroms. Elution volumes of about 15-19 ml correspondto particles of Stokes' diameters of 50-120 Angstroms, which is the sizerange of human HDL.

12.2. SUPEROSE 6 GEL FILTRATION CHROMATOGRAPHY OF HUMAN HDL

Human HDL₂ was prepared as follows: 300 ml frozen human plasma (MannheimBlutspendzentrale #1185190) was thawed, adjusted to density 1.25 withsolid potassium bromide, and centrifuged for 45 hours at 40,000 PRMusing a Ti45 rotor (Beckman) at 20° C. The floating layer was collected,dialyzed against distilled water, adjusted to density 1.07 with solidpotassium bromide, and centrifuged as described above for 70 hours. Thebottom layer (at a level of one cm above the tube bottom) was collected,brought to 0.01% sodium azide, and stored at 4° C. for 4 days untilchromatography. 20 μl of the HDL₂ was loaded onto a Pharmacia Superose 6FPLC gel filtration chromatography system using 0.9% NaCl as columneluate. The column flow rate was 0.5 ml/min. The column eluate wasmonitored by absorbance or scattering of light of wavelength 254 nm. Aseries of proteins of known molecular weight and Stokes' diameter wereused as standards to calibrate the column for the calculation of Stokes'diameters of the particles (Pharmacia Gel Filtration Calibration KitInstruction Manual, Pharmacia Laboratory Separation, Piscataway, N.J.,revised April 1985). The HDL eluted with a retention volume of 14.8 ml,corresponding to a Stokes' diameter of 108 nm.

13. EXAMPLE: PREPARATION OF ANTIBODIES

Peptides 4 or 8 were conjugated to keyhole limpet hemocyanine (KLH; 1 mgpeptide to 10 mg KLH). The KLH conjugate (LMG) was suspended in completeFreund's adjuvant and injected into rabbits at time 0, and boosted with0.25 mg KLH conjugate at 4 weeks and again at 5 weeks. Pre-bleeds andsix week post-bleeds were tested for antibody titer against authenticantigen by ELISA.

The production bleeds were pooled from 2 rabbits each. Antibodiesdirected exclusively against the peptide antigens were isolated asfollows:

1. Free peptide was attached to cyanogen bromide activated Sepharose 4B(Pharmacia) according to the manufacturer's protocol.

2. The antisera was preabsorbed on a column of irrelevant peptides andon columns of irrelevant human and mouse serum proteins.

3. The pre-absorbed antisera was passed through the correspondingpeptide column (see point 1).

4. The columns were washed with 0.1 M borate buffered saline (pH 8.2)and the bound antibodies were eluted using a low pH gradient step frompH 4.0 to pH 3.0 to pH 2.0 (0.1 M glycine buffer) and finally with 0.1 MHCl.

5. The eluted material was neutralized with excess borate saline,concentrated by ultrafiltration (Amicon, YM30) and dialyzed againstborate saline.

6. The protein concentration was determined by absorbance at 280 nm.

The resulting antibodies were tested for species specificity usingpurified human ApoA-I or purified mouse ApoA-I in a direct ELISA bindingassay. The human and murine antibodies were specific for human ApoA-I,and demonstrated minimal cross-activity.

The invention is not to be limited in scope by the specific embodimentsdescribed which are intended as single illustrations of individualaspects of the invention, and functionally equivalent methods andcomponents are within the scope of the invention. Indeed variousmodifications of the invention, in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and accompanying drawings. Such modifications areintended to fall within the scope of the appended claims.

All references cited herein are incorporated herein by reference for allpurposes.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 258                                         - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 16                                                              (D) OTHER INFORMATION: - #Xaa = Naphthylalanine                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu        Xaa                                                                              1               5  - #                10  - #                15              - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Gly Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys Lys                                                          20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Trp       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #Xaa = D-Pro                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Xaa Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys Lys                                                          20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Pro Val Leu Asp Leu Phe Lys Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Pro Val Leu Asp Leu Phe Arg Glu Leu Gly As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #Xaa = Naphthylalanine                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Xaa Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Pro Val Leu Asp Leu Phe Lys Glu Leu Leu Gl - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Gly Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - Gly Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 20                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 22                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Xaa Gln Xaa Leu Xaa                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Trp As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - Pro Val Leu Asp Leu Leu Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - Pro Val Leu Glu Leu Phe Lys Glu Leu Leu Gl - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - Gly Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #Xaa = D-Pro                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - Xaa Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - Pro Val Leu Asp Leu Phe Arg Glu Gly Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #Xaa = D-Pro                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - Xaa Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Gly       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - Pro Leu Leu Glu Leu Phe Lys Glu Leu Leu Gl - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - Pro Val Leu Asp Phe Phe Arg Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Leu       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #Xaa = Naphthylalanine                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Xaa Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Trp Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - Ala Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...22                                                          (D) OTHER INFORMATION: - #N-terminal dansylated peptide              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - Pro Val Leu Asp Leu Phe Leu Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - Xaa Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - Pro Val Leu Asp Leu Phe Arg Glu Lys Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 5                                                               (D) OTHER INFORMATION: - #Xaa = Naphthylalanine                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - Pro Val Leu Asp Xaa Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - Pro Val Leu Asp Trp Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - Pro Leu Leu Glu Leu Leu Lys Glu Leu Leu Gl - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - Pro Val Leu Asp Leu Phe Arg Glu Trp Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Trp Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Leu Lys Ala       1               5  - #                10  - #                15               - - Leu Lys Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - Pro Val Leu Asp Leu Phe Asn Glu Leu Leu Ar - #g Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - Pro Val Leu Asp Leu Trp Arg Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Trp Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Tr - #p Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...22                                                          (D) OTHER INFORMATION: - #All genetically encoded amino acids                      are in - #the D-configuration                                            (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - Pro Val Leu Asp Leu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Gl - #u Gly Leu Glu Ala Leu       1               5  - #                10  - #                15               - - Lys Gln Lys Leu Lys                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #Xaa = D-Pro                                         (A) NAME/KEY: Other                                                           (B) LOCATION: 2                                                               (D) OTHER INFORMATION: - #Xaa = D-Val                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - Xaa Xaa Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - Pro Val Leu Asp Leu Phe Arg Asn Leu Leu Gl - #u Lys Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Glu Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Tr - #p Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - Pro Val Leu Asp Leu Phe Trp Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - Pro Val Trp Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - Val Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Trp Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - Pro Leu Phe Arg Glu Leu Leu Asn Glu Leu Le - #u Glu Ala Leu Lys Gln       1               5  - #                10  - #                15               - - Lys Leu Lys                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Lys Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - Pro Val Leu Asp Leu Phe Arg Asn Leu Leu Gl - #u Glu Leu Leu Lys Ala       1               5  - #                10  - #                15               - - Leu Glu Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - Leu Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - Pro Val Leu Asp Glu Phe Arg Trp Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - Pro Val Leu Asp Glu Trp Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - Pro Val Leu Asp Phe Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - Pro Trp Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 12                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - Val Leu Asp Glu Phe Arg Glu Lys Leu Asn Gl - #u Xaa Leu Glu Ala Leu       1               5  - #                10  - #                15               - - Lys Gln Lys Leu Lys                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - Pro Val Leu Asp Leu Phe Arg Asn Leu Leu Gl - #u Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - Val Leu Asp Leu Phe Arg Glu Leu Leu Asn Gl - #u Leu Leu Glu Ala Leu       1               5  - #                10  - #                15               - - Lys Gln Lys Leu Lys                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - Pro Val Leu Asp Glu Phe Arg Glu Leu Leu Ly - #s Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - Pro Val Leu Asp Glu Phe Arg Lys Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - Pro Val Leu Asp Glu Phe Arg Glu Leu Leu Ty - #r Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:71:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Leu Xaa Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:72:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Xaa Leu Trp Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:73:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                              - - Pro Val Leu Asp Glu Phe Trp Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:74:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                              - - Pro Val Leu Asp Lys Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:75:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Glu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:76:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                              - - Pro Val Leu Asp Glu Phe Arg Glu Leu Leu Ph - #e Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:77:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Lys Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:78:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                              - - Pro Val Leu Asp Glu Phe Arg Asp Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:79:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                              - - Pro Val Leu Asp Glu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:80:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                              - - Pro Val Leu Asp Leu Phe Glu Arg Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:81:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Trp Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:82:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 11                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                              - - Leu Asp Glu Phe Arg Glu Lys Leu Asn Glu Xa - #a Leu Glu Ala Leu Lys       1               5  - #                10  - #                15               - - Gln Lys Leu Lys                                                                      20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:83:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Trp Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:84:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:85:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                              - - Pro Leu Asp Leu Phe Arg Glu Leu Leu Asn Gl - #u Leu Leu Glu Ala Leu       1               5  - #                10  - #                15               - - Lys Gln Lys Leu Lys                                                                  20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:86:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                              - - Pro Val Leu Glu Leu Phe Glu Arg Leu Leu As - #p Glu Leu Leu Asn Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:87:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...22                                                          (D) OTHER INFORMATION: - #All amino acids are in the                               D-configurat - #ion                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                              - - Pro Leu Leu Glu Leu Leu Lys Glu Leu Leu Gl - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:88:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                              - - Pro Val Leu Asp Lys Phe Arg Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:89:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Trp Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:90:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                              - - Asp Glu Phe Arg Glu Lys Leu Asn Glu Xaa Le - #u Glu Ala Leu Lys Gln       1               5  - #                10  - #                15               - - Lys Leu Lys                                                               - -  - - (2) INFORMATION FOR SEQ ID NO:91:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                              - - Pro Val Leu Asp Glu Phe Arg Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:92:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                              - - Pro Val Leu Asp Glu Phe Arg Glu Leu Tyr As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:93:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Lys Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:94:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                              - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Ala Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:95:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Leu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:96:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...22                                                          (D) OTHER INFORMATION: - #All genetically encoded amino acids                      are in - #the D-configuration                                            (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:97:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu           1               5  - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:98:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                              - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Glu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:99:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                              - - Lys Leu Lys Gln Lys Leu Ala Glu Leu Leu Gl - #u Asn Leu Leu Glu Arg       1               5  - #                10  - #                15               - - Phe Leu Asp Leu Val Pro                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:100:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...22                                                          (D) OTHER INFORMATION: - #All amino acids are in the                               D-configurat - #ion                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:101:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Trp Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:102:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                             - -  Pro Val Leu Asp Leu Phe Arg Glu Leu Leu - #Asn Leu Xaa Leu Glu Ala        1               5 - #                 10 - #                 15              - -  Leu Lys Glu Lys Leu Lys                                                              20                                                                - -  - - (2) INFORMATION FOR SEQ ID NO:103:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                             - - Pro Val Leu Asp Glu Phe Arg Glu Leu Leu As - #n Glu Glu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:104:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                             - - Pro Leu Leu Asn Glu Leu Leu Glu Ala Leu Ly - #s Gln Lys Leu Lys           1               5  - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:105:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                             - - Pro Ala Ala Asp Ala Phe Arg Glu Ala Ala As - #n Glu Ala Ala Glu Ala       1               5  - #                10  - #                15               - - Ala Lys Gln Lys Ala Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:106:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                             - - Pro Val Leu Asp Leu Phe Arg Glu Lys Leu As - #n Glu Glu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:107:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...22                                                          (D) OTHER INFORMATION: - #All amino acids are in the                               D-configurat - #ion                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                             - - Lys Leu Lys Gln Lys Leu Ala Glu Leu Leu Gl - #u Asn Leu Leu Glu Arg       1               5  - #                10  - #                15               - - Phe Leu Asp Leu Val Pro                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:108:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                             - - Pro Val Leu Asp Leu Phe Arg Trp Leu Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:109:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Arg Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:110:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Xaa Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:111:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Tr - #p Glu Xaa Trp Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:112:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Ser Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:113:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Pro Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:114:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Met Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:115:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                             - - Pro Lys Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:116:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                             - - Pro His Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:117:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                             - - Pro Glu Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:118:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 13                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Xaa Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Glu Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:119:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Glu Leu Glu Ala       1               5  - #                10  - #                15               - - Xaa Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:120:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 16                                                              (D) OTHER INFORMATION: - #Xaa = Aib                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Glu Leu Glu Xaa       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:121:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Glu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Trp Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:122:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:                             - - Pro Val Leu Asp Glu Phe Arg Glu Lys Leu As - #n Glu Glu Leu Glu Trp       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:123:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:                             - - Gln Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:124:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 20                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 22                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:                             - - Pro Val Leu Asp Leu Phe Xaa Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Xaa Gln Xaa Leu Xaa                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:125:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:                             - - Asn Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:126:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Gly Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:127:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Leu       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:128:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Phe       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:129:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:                             - - Pro Val Leu Glu Leu Phe Asn Asp Leu Leu Ar - #g Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:130:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:                             - - Pro Val Leu Glu Leu Phe Asn Asp Leu Leu Ar - #g Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:131:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:                             - - Pro Val Leu Glu Leu Phe Lys Glu Leu Leu As - #n Glu Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Arg Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:132:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Asn Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:133:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:                             - - Pro Val Leu Glu Leu Phe Glu Arg Leu Leu Gl - #u Asp Leu Leu Gln Ala       1               5  - #                10  - #                15               - - Leu Asn Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:134:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:                             - - Pro Val Leu Glu Leu Phe Glu Arg Leu Leu Gl - #u Asp Leu Leu Lys Ala       1               5  - #                10  - #                15               - - Leu Asn Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:135:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:                             - - Asp Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:136:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:                             - - Pro Ala Leu Glu Leu Phe Lys Asp Leu Leu Gl - #n Glu Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:137:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #Xaa = Naphthylalanine                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Xaa Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:138:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Trp       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:139:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Trp As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:140:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 20                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 22                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Xaa Gln Xaa Leu Xaa                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:141:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:                             - - Pro Val Leu Asp Phe Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:142:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:                             - - Pro Val Leu Glu Leu Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:143:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...22                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated peptide                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Gly Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Lys Gln Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:144:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #Xaa = D-Pro                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:                             - - Xaa Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:145:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:145:                             - - Gly Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:146:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:146:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:147:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:147:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Phe Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:148:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:148:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Gly Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:149:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:149:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Trp Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:150:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:150:                             - - Pro Leu Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:151:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:151:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Gly Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:152:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:152:                             - - Pro Val Phe Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:153:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:153:                             - - Ala Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:154:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:154:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Gly Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:155:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:155:                             - - Pro Val Leu Glu Leu Phe Leu Asn Leu Trp Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:156:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:156:                             - - Pro Val Leu Glu Leu Phe Leu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:157:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:157:                             - - Pro Val Leu Glu Phe Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:158:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:158:                             - - Pro Val Leu Glu Leu Phe Leu Asn Leu Leu Gl - #u Arg Leu Leu Asp Trp       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:159:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:159:                             - - Pro Val Leu Asp Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:160:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:160:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Trp       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:161:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:161:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:162:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:162:                             - - Pro Val Leu Glu Leu Phe Glu Asn Trp Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:163:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:163:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Trp Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:164:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:164:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Trp Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:165:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:165:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Leu       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:166:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:166:                             - - Pro Val Leu Glu Leu Phe Leu Asn Leu Leu Gl - #u Lys Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:167:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:167:                             - - Pro Val Leu Glu Leu Phe Glu Asn Gly Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:168:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:168:                             - - Pro Val Leu Glu Leu Phe Glu Gln Leu Leu Gl - #u Lys Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:169:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:169:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Lys Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:170:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 12                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 19                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 20                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 22                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:170:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Xaa Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Xaa Xaa Leu Xaa                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:171:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:171:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Lys Leu Leu Asp Leu       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:172:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:172:                             - - Pro Val Leu Glu Leu Phe Leu Asn Leu Leu Gl - #u Arg Leu Gly Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:173:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:173:                             - - Pro Val Leu Asp Leu Phe Asp Asn Leu Leu As - #p Arg Leu Leu Asp Leu       1               5  - #                10  - #                15               - - Leu Asn Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:174:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...22                                                          (D) OTHER INFORMATION: - #All amino acids are in the                               D-configurat - #ion                                             - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:174:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:175:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:175:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Glu Leu       1               5  - #                10  - #                15               - - Leu Asn Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:176:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:176:                             - - Pro Val Leu Glu Leu Trp Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:177:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:177:                             - - Gly Val Leu Glu Leu Phe Leu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:178:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:178:                             - - Pro Val Leu Glu Leu Phe Asp Asn Leu Leu Gl - #u Lys Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Arg                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:179:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:179:                             - - Pro Val Leu Glu Leu Phe Asp Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:180:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:180:                             - - Pro Val Leu Glu Leu Phe Asp Asn Leu Leu As - #p Lys Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Arg                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:181:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:181:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Trp Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:182:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:182:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Lys Leu Leu Glu Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:183:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:183:                             - - Pro Leu Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Lys Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:184:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:184:                             - - Pro Val Leu Glu Leu Phe Leu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Trp Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:185:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 19                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 20                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 22                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:185:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Xaa Xaa Leu Xaa                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:186:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:186:                             - - Pro Val Leu Glu Leu Phe Glu Gln Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:187:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:187:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Asn Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:188:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:188:                             - - Pro Val Leu Glu Leu Phe Glu Asn Leu Leu As - #p Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:189:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:189:                             - - Asp Val Leu Glu Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Lys                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:190:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:190:                             - - Pro Val Leu Glu Phe Trp Asp Asn Leu Leu As - #p Lys Leu Leu Asp Ala       1               5  - #                10  - #                15               - - Leu Gln Lys Lys Leu Arg                                                              20                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:191:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:191:                             - - Pro Val Leu Asp Leu Leu Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:192:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:192:                             - - Pro Val Leu Asp Leu Phe Lys Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:193:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:193:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:194:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:194:                             - - Pro Val Leu Glu Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:195:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:195:                             - - Pro Val Leu Glu Leu Phe Lys Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:196:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:196:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Asn Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:197:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:197:                             - - Pro Leu Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:198:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:198:                             - - Gly Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:199:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:199:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Trp Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:200:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:200:                             - - Asn Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:201:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:201:                             - - Pro Leu Leu Asp Leu Phe Lys Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:202:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:202:                             - - Pro Ala Leu Glu Leu Phe Lys Asp Leu Leu Gl - #u Glu Leu Arg Gln Lys       1               5  - #                10  - #                15               - - Leu Arg                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:203:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:203:                             - - Ala Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:204:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:204:                             - - Pro Val Leu Asp Phe Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:205:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:205:                             - - Pro Val Leu Asp Leu Phe Arg Glu Trp Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:206:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:206:                             - - Pro Leu Leu Glu Leu Leu Lys Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:207:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:207:                             - - Pro Val Leu Glu Leu Leu Lys Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:208:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:208:                             - - Pro Ala Leu Glu Leu Phe Lys Asp Leu Leu Gl - #u Glu Leu Arg Gln Arg       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:209:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:209:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:210:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:210:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:211:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                                   (A) NAME/KEY: Other                                                           (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 16                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 18                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:211:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Xaa Gln Xaa       1               5  - #                10  - #                15               - - Leu Xaa                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:212:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                                   (A) NAME/KEY: Other                                                           (B) LOCATION: 7                                                               (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 14                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                           (A) NAME/KEY: Other                                                           (B) LOCATION: 16                                                              (D) OTHER INFORMATION: - #Xaa = Orn                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:212:                             - - Pro Val Leu Asp Leu Phe Xaa Glu Leu Leu Gl - #u Glu Leu Xaa Gln Xaa       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:213:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:213:                             - - Pro Ala Leu Glu Leu Phe Lys Asp Leu Leu Gl - #u Glu Phe Arg Gln Arg       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:214:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                                   (A) NAME/KEY: Other                                                           (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #D-configuration of Pro                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:214:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:215:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:215:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Trp Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:216:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:216:                             - - Pro Val Leu Glu Leu Phe Lys Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:217:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:217:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Leu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:218:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:218:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Leu Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:219:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:219:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Trp Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:220:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:220:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Gln Lys Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:221:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:221:                             - - Asp Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:222:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:222:                             - - Pro Val Leu Asp Ala Phe Arg Glu Leu Leu Gl - #u Ala Leu Leu Gln Leu       1               5  - #                10  - #                15               - - Lys Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:223:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:223:                             - - Pro Val Leu Asp Ala Phe Arg Glu Leu Leu Gl - #u Ala Leu Ala Gln Leu       1               5  - #                10  - #                15               - - Lys Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:224:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:224:                             - - Pro Val Leu Asp Leu Phe Arg Glu Gly Trp Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:225:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:225:                             - - Pro Val Leu Asp Ala Phe Arg Glu Leu Ala Gl - #u Ala Leu Ala Gln Leu       1               5  - #                10  - #                15               - - Lys Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:226:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:226:                             - - Pro Val Leu Asp Ala Phe Arg Glu Leu Gly Gl - #u Ala Leu Leu Gln Leu       1               5  - #                10  - #                15               - - Lys Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:227:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:227:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Gly Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:228:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:228:                             - - Pro Val Leu Asp Leu Phe Arg Glu Gly Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:229:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:229:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Gly Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:230:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:230:                             - - Pro Val Leu Glu Leu Phe Glu Arg Leu Leu Gl - #u Asp Leu Gln Lys Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:231:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:231:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Lys Leu Glu Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:232:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:232:                             - - Pro Leu Leu Glu Leu Phe Lys Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - - Leu Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:233:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:233:                             - - This sequence has been intentionally skipped                              - -  - - (2) INFORMATION FOR SEQ ID NO:234:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:234:                             - - This sequence has been intentionally skipped                              - -  - - (2) INFORMATION FOR SEQ ID NO:235:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:235:                             - - This sequence has been intentionally skipped                              - -  - - (2) INFORMATION FOR SEQ ID NO:236:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:236:                             - - This sequence has been intentionally skipped                              - -  - - (2) INFORMATION FOR SEQ ID NO:237:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:237:                             - - Leu Asp Asp Leu Leu Gln Lys Trp Ala Glu Al - #a Phe Asn Gln Leu Leu       1               5  - #                10  - #                15               - - Lys Lys                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:238:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:238:                             - - Glu Trp Leu Lys Ala Phe Tyr Glu Lys Val Le - #u Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:239:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:239:                             - - Glu Trp Leu Glu Ala Phe Tyr Lys Lys Val Le - #u Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:240:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:240:                             - - Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Al - #a Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Ala Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:241:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:241:                             - - Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ph - #e Glu Lys Phe Lys Glu       1               5  - #                10  - #                15               - - Phe Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:242:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:242:                             - - Gly Ile Lys Lys Phe Leu Gly Ser Ile Trp Ly - #s Phe Ile Lys Ala Phe       1               5  - #                10  - #                15               - - Val Gly                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:243:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:243:                             - - Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Al - #a Glu Lys Phe Lys Glu       1               5  - #                10  - #                15               - - Ala Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:244:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:244:                             - - Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Al - #a Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Ala Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:245:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:245:                             - - Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ph - #e Glu Lys Phe Lys Glu       1               5  - #                10  - #                15               - - Phe Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:246:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:246:                             - - Glu Trp Leu Glu Ala Phe Tyr Lys Lys Val Le - #u Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:247:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:247:                             - - Asp Trp Phe Lys Ala Phe Tyr Asp Lys Phe Ph - #e Glu Lys Phe Lys Glu       1               5  - #                10  - #                15               - - Phe Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:248:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:248:                             - - Glu Trp Leu Lys Ala Phe Tyr Glu Lys Val Le - #u Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:249:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:249:                             - - Glu Trp Leu Lys Ala Glu Tyr Glu Lys Val Gl - #u Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:250:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:250:                             - - Glu Trp Leu Lys Ala Glu Tyr Glu Lys Val Le - #u Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:251:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...18                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:251:                             - - Glu Trp Leu Lys Ala Phe Tyr Lys Lys Val Le - #u Glu Lys Leu Lys Glu       1               5  - #                10  - #                15               - - Leu Phe                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:252:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...15                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:252:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Gln Lys Leu Lys           1               5  - #                10  - #                15               - -  - - 2) INFORMATION FOR SEQ ID NO:253:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...16                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:253:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Glu Leu Lys Gln Lys       1               5  - #                10  - #                15               - -  - - 2) INFORMATION FOR SEQ ID NO:254:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...16                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:254:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Lys Leu Lys Gln Lys       1               5  - #                10  - #                15               - -  - - 2) INFORMATION FOR SEQ ID NO:255:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...15                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:255:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Lys Leu Gln Lys           1               5  - #                10  - #                15               - -  - - 2) INFORMATION FOR SEQ ID NO:256:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...16                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:256:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu Gl - #u Ala Leu Lys Gln Lys       1               5  - #                10  - #                15               - -  - - 2) INFORMATION FOR SEQ ID NO:257:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...16                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:257:                             - - Pro Val Leu Asp Leu Phe Glu Asn Leu Leu Gl - #u Arg Leu Lys Gln Lys       1               5  - #                10  - #                15               - -  - - 2) INFORMATION FOR SEQ ID NO:258:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: None                                              - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Other                                                           (B) LOCATION: 1...16                                                          (D) OTHER INFORMATION: - #N-terminal acetylated and                                C-terminal - #amidated                                          - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:258:                             - - Pro Val Leu Asp Leu Phe Arg Glu Leu Leu As - #n Glu Leu Lys Gln Lys       1               5  - #                10  - #                15             __________________________________________________________________________

What is claimed is:
 1. An ApoA-I agonist compound comprising:(i) a 22 to29-residue peptide or peptide analogue which forms an amphipathicα-helix in the presence of lipids and which comprises formula (I): Z₁-X₁ -X₂ -X₃ -X₄ -X₅ -X₆ -X₇ -X₈ -X₉ -X₁₀ -X₁₁ -X₁₂ -X₁₃ -X₁₄ -X₁₅ -X₁₆-X₁₇ -X₁₈ -X₁₉ -X₂₀ -X₂₁ -X₂₂ -X₂₃ -Z₂ or a pharmaceutically acceptablesalt thereof, wherein: X₁ is Pro (P), Ala (A), Gly (G), Gln (Q), Asn(N), Asp (D) or D-Pro (p); X₂ is an aliphatic residue; X₃ is Leu (L) orPhe (F); X₄ is an acidic residue; X₅ is Leu (L) or Phe (F); X₆ is Leu(L) or Phe (F); X₇ is a hydrophilic residue; X₈ is an acidic or a basicresidue; X₉ is Leu (L) or Gly (G); X₁₀ is Leu (L), Trp (W) or Gly (G);X₁₁ is a hydrophilic residue; X₁₂ is a hydrophilic residue; X₁₃ is Gly(G) or an aliphatic residue; X₁₄ is Leu (L), Trp (W), Gly (G) or Nal;X₁₅ is a hydrophilic residue; X₁₆ is a hydrophobic residue; X₁₇ is ahydrophobic residue; X₁₈ is Gln (Q), Asn (N) or a basic residue; X₁₉ isGln (Q), Asn (N) or a basic residue; X₂₀ is a basic residue; X₂₁ is analiphatic residue; X₂₂ is a basic residue; X₂₃ is absent or a basicresidue; Z₁ is H₂ N-- or RC(O)NH--; Z₂ is --C(O)NRR, --C(O)OR or--C(O)OH or a salt thereof; each R is independently --H, (C₁ -C₆) alkyl,(C₁ -C₆) alkenyl, (C₁ -C₆) alkynyl, (C₅ -C₂₀) aryl, (C₆ -C₂₆) alkaryl,5-20 membered heteroaryl, 6-26 membered alkheteroaryl or a 1 to7-residue peptide or peptide analogue in which one or more bonds betweenresidues 1-7 are independently a substituted amide, and isostere of anamide or an amide mimetic; and each "--" between residues X₁ through X₂₃independently designates an amide linkage, a substituted amide linkage,an isostere of an amide or an amide mimetic; or (ii) an altered form offormula (I) in which at least one of residues X₁, X₂, X₃, X₄, X₅, X₆,X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁,X₂₂ or X₂₃ is conservatively substituted with another residue.
 2. TheApoA-I agonist compound of claim 1 which exhibits at least about 38%LCAT-activation activity as compared with human ApoA-I.
 3. The ApoA-Iagonist compound of claim 1 which is the altered form of formula (I). 4.The ApoA-I agonist compound of claim 3 in which residues X₂, X₃, X₅, X₆,X₉, X₁₀, X₁₃, X₁₄, X₁₆, X₁₇ and X₂₁ are fixed according to formula (I)and at least one of residues X₁, X₄, X₇, X₈, X₁₁, X₁₂, X₁₅, X₁₈, X₁₉,X₂₀ and X₂₂ is conservatively substituted with another residue.
 5. TheApoA-I agonist compound of claim 4 in which:X₁ is Pro (P), D-Pro (p),Gly (G) or Ala (A); X₂ is Ala (A), Leu (L) or Val (V); X₃ is Leu (L) orPhe (F); X₅ is Leu (L) or Phe (F); X₆ is Leu (L) or Phe (F); X₉ is Leu(L) or Gly (G); X₁₀ is Leu (L), Trp (W) or Gly (G); X₁₃ is Leu (L), Gly(G) or Aib; X₁₄ is Leu, Nal, Trp (W) or Gly (G); X₁₆ is Ala (A), Nal,Trp (W), Gly (G), Leu (L) or Phe (F); X₁₇ is Leu (L), Gly (G) or Nal;and X₂₁ is Leu (L).
 6. The ApoA-I agonist compound of claim 3 in whichresidues X₄, X₇, X₈, X₁₁, X₁₂, X₁₅, X₁₈, X₁₉ and X₂₂ are fixed accordingto formula (I) and at least one of residues X₂, X₃, X₅, X₆, X₉, X₁₀,X₁₃, X₁₄, X₁₆, X₁₇, X₂₀ and X₂₁ is conservatively substituted withanother residue.
 7. The ApoA-I agonist compound of claim 6 in which:X₄is Asp (D) or Glu (E); X₇ is Lys (K), Arg (R) or Orn; X₈ is Asp (D) orGlu (E); X₁₁ is Asn (N) or Gln (Q); X₁₂ is Glu (E) or Asp (D); X₁₅ isAsp (D) or Glu (E); X₁₈ is Gln (Q), Asn (N), Lys (K) or Orn; X₁₉ is Gln(Q), Asn (N), Lys (K) or Orn; X₂₀ is Lys (K) or Orn; X₂₂ is Lys (K) orOrn; and X₂₃ is absent or Lys (K).
 8. The ApoA-I agonist compound ofclaim 7 in which X₃ is Leu (L) or Phe (F), X₆ is Phe (F), X₉ is Leu (L)or Gly (G), X₁₀ is Leu (L) or Trp (W) or Gly (G).
 9. The ApoA-I agonistcompound of claim 5 or 7 in which the conservatively substitutingresidue is classified within a same sub-category as the conservativelysubstituted residue.
 10. The ApoA-I agonist compound of claim 1 which isa 22-23 residue peptide or peptide analogue according to formula (I).11. The ApoA-I agonist compound of claim 10 in which:the "--" betweenresidues X₁ through X₂₃ designates --C(O)NH--; Z₁ is H₂ N--; and Z₂ is--C(O)OH or a salt thereof.
 12. The ApoA-I agonist compound of claim 11,in which:X₁ is Pro (P), Ala (A), Gly (G), Asn (N), Gln (Q), Asp (D) orD-Pro (p); X₂ is Ala (A), Val (V) or Leu (L); X₃ is Leu (L) or Phe (F);X₄ is Asp (D) or Glu (E); X₅ is Leu (L) or Phe (F); X₆ is Leu (L) or Phe(F); X₇ is Lys (K), Arg (R) or Orn; X₈ is Asp (D) or Glu (E); X₉ is Leu(L) or Gly (G); X₁₀ is Leu (L), Trp (W) or Gly (G); X₁₁ is Asn (N) orGln (Q); X₁₂ is Glu (E) or Asp (D); X₁₃ is Gly (G), Leu (L) or Aib; X₁₄is Leu (L), Nal, Trp (W) or Gly (G); X₁₅ is Asp (D) or Glu (E); X₁₆ isAla (A), Nal, Trp (W), Leu (L), Phe (F) or Gly (G); X₁₇ is Gly (G), Leu(L) or Nal; X₁₈ is Gln (Q), Asn (N), Lys (K) or Orn; X₁₉ is Gln (Q), Asn(N), Lys (K) or Orn; X₂₀ is Lys (K) or Orn; X₂₁ is Leu (L); X₂₂ is Lys(K) or Orn; and X₂₃ is absent or Lys (K).
 13. The ApoA-I agonistcompound of claim 12, in which X₂₃ is absent.
 14. The ApoA-I agonistcompound of claim 11 or 12, in which one of X₁₈ or X₁₉ is Gln (Q) or Asn(N) and the other of X₁₈ or X₁₉ is Lys (K) or Orn.
 15. The ApoA-Iagonist compound of claim 13 in which each of X₉, X₁₀, X₁₃, X₁₄, X₁₆ andX₁₇ is other than Gly (G).
 16. The ApoA-I agonist compound of claim 13in which one of X₉, X₁₀, X₁₃, X₁₄, X₁₆ and X₁₇ is Gly (G) and the othersare other than Gly (G).
 17. The ApoA-I agonist compound of claim 1 whichis selected from the group consisting of:

    ______________________________________                                        peptide 1                                                                              PVLDLFRELLNELLEZLKQKLK                                                                          (SEQ ID NO:1)                                      peptide 2          GVLDLFRELLNELLEALKQKLKK (SEQ ID NO:2)                         - peptide 3    PVLDLFRELLNELLEWLKQKLK  (SEQ ID NO:3)                          - peptide 4    PVLDLFRELLNELLEALKQKLK  (SEQ ID NO:4)                          - peptide 5    pVLDLFRELLNELLEALKQKLKK (SEQ ID NO:5)                          - peptide 6    PVLDLFRELLNEXLEALKQKLK  (SEQ ID NO:6)                          - peptide 7    PVLDLFKELLNELLEALKQKLK  (SEQ ID NO:7)                          - peptide 8    PVLDLFRELLNEGLEALKQKLK  (SEQ ID NO:8)                          - peptide 9    PVLDLFRELGNELLEALKQKLK  (SEQ ID NO:9)                          - peptide 10   PVLDLFRELLNELLEAZKQKLK  (SEQ ID NO:10)                         - peptide 11   PVLDLFKELLQELLEALKQKLK  (SEQ ID NO:11)                         - peptide 12   PVLDLFRELLNELLEAGKQKLK  (SEQ ID NO:12)                         - peptide 13   GVLDLFRELLNEGLEALKQKLK  (SEQ ID NO:13)                         - peptide 14   PVLDLFRELLNELLEALOQOLO  (SEQ ID NO:14)                         - peptide 15   PVLDLFRELWNELLEALKQKLK  (SEQ ID NO:15)                         - peptide 16   PVLDLLRELLNELLEALKQKLK  (SEQ ID NO:16)                         - peptide 17   PVLELFKELLQELLEALKQKLK  (SEQ ID NO:17)                         - peptide 18   GVLDLFRELLNELLEALKQKLK  (SEQ ID NO:18)                         - peptide 19   pVLDLFRELLNEGLEALKQKLK  (SEQ ID NO:19)                         - peptide 20   PVLDLFREGLNELLEALKQKLK  (SEQ ID NO:20)                         - peptide 21   pVLDLFRELLNELLEALKQKLK  (SEQ ID NO:21)                         - peptide 22   PVLDLFRELLNELLEGLKQKLK  (SEQ ID NO:22)                         - peptide 23   PLLELFKELLQELLEALKQKLK  (SEQ ID NO:23)                         - peptide 24   PVLDLFRELLNELLEALQKKLK  (SEQ ID NO:24)                         - peptide 25   PVLDFFRELLNEXLEALKQKLK  (SEQ ID NO:25)                         - peptide 26   PVLDLFRELLNELLELLKQKLK  (SEQ ID NO:26)                         - peptide 27   PVLDLFRELLNELZEALKQKLK  (SEQ ID NO:27)                         - peptide 28  PVLDLFRELLNELWEALKQKLK   (SEQ ID NO:28)                         - peptide 29  AVLDLFRELLNELLEALKQKLK   (SEQ ID NO:29)                         - peptide 123 QVLDLFRELLNELLEALKQKLK   (SEQ ID NO:123)                        - peptide 124 PVLDLFQELLNELLEALOQOLO   (SEQ ID NO:124)                        - peptide 125 NVLDLFRELLNELLEALKQKLK   (SEQ ID NO:125)                        - peptide 126 PVLDLFRELLNELGEALKQKLK   (SEQ ID NO:126)                        - peptide 127 PVLDLFRELLNELLELLKQKLK   (SEQ ID NO:127)                        - peptide 128 PVLDLFRELLNELLEFLKQKLK   (SEQ ID NO:128)                        - peptide 129 PVLELFNDLLRELLEALQKKLK   (SEQ ID NO:129)                        - peptide 130 PVLELFNDLLRELLEALKQKLK   (SEQ ID NO:130)                        - peptide 131 PVLELFKELLNELLDALRQKLK   (SEQ ID NO:131)                        - peptide 132 PVLDLFRELLENLLEALQKKLK   (SEQ ID NO:132)                        - peptide 133 PVLELFERLLEDLLQALNKKLK   (SEQ ID NO:133)                        - peptide 134 PVLELFERLLEDLLKALNQKLK   (SEQ ID NO:134)                        - peptide 135 DVLDLFRELLNELLEALKQKLK   (SEQ ID NO:135)                        - peptide 136 PALELFKDLLQELLEALKQKLK   (SEQ ID NO:136)                        - peptide 137 PVLDLFRELLNEGLEAZKQKLK   (SEQ ID NO:137)                        - peptide 138 PVLDLFRELLNEGLEWLKQKLK   (SEQ ID NO:138)                        - peptide 139 PVLDLFRELWNEGLEALKQKLK   (SEQ ID NO:139)                        - peptide 140 PVLDLFRELLNEGLEALOQOLO   (SEQ ID NO:140)                        - peptide 141 PVLDFFRELLNEGLEALKQKLK   (SEQ ID NO:141)                        -   and                                                                       - peptide 142 PVLELFRELLNEGLEALKQKLK   (SEQ ID NO:142);                    ______________________________________                                    

and N-terminal acylated and/or C-terminal amidated or esterified formsthereof, wherein X is Aib; Z is Nal; and O is Orn.
 18. A pharmaceuticalcomposition comprising an ApoA-I agonist compound and a pharmaceuticallyacceptable carrier, excipient or diluent, wherein the ApoA-I agonistcompound is a peptide or peptide analogue according to claim
 1. 19. Thepharmaceutical composition of claim 18 in which the ApoA-I agonistcompound is a peptide according to claim
 10. 20. The pharmaceuticalcomposition of claim 18 in which the ApoA-I agonist compound is apeptide according to claim
 11. 21. The pharmaceutical composition ofclaim 18 in which the ApoA-I agonist compound is a peptide according toclaim
 12. 22. The pharmaceutical composition of claim 18 in which theApoA-I agonist compound is a peptide according to claim
 17. 23. TheApoA-I agonist compound of claim 1 in which Z₁ is RC(O)NH-- and Z₂ is--C(O)NRR or --C(O)OR.
 24. The ApoA-I agonist compound of claim 10 inwhich X₂₃ is absent and each "--" between residues X₁ through X₂₂designates --C(O)NH--.
 25. The ApoA-I agonist compound of claim 24 inwhich X₇ is a basic amino acid, Asn (N) or Glu (E); X₈ is an acidicamino acid or Arg (R); X₁₂ is an acidic amino acid or Asn (N); and/orX₁₅ is an acidic amino acid, Gln (Q) or Lys (K).
 26. The ApoA-I agonistcompound of claim 24 in which:X₁ is Pro (P), Gly (G), Ala (A), Gln (Q),Asn (N), Asp (D) or D-Pro (p); X₂ is Ala (A), Val (V) or Leu (L); X₄ isAsp (D) or Glu (E); X₇ is Lys (K), Arg (R), Orn, Asn (N) or Glu (E); X₈is Asp (D), Arg (R) or Glu (E); X₁₁ is Asn (N), Gln (Q), Glu (E) or Arg(R); X₁₂ is Asp (D), Glu (E) or Asn (N); X₁₃ is Leu (L), Gly (G) or Aib;X₁₅ is Asp (D), Glu (E), Gln (Q) or Lys (K); X₁₆ is Ala (A), Trp (W),Gly (G), Leu (L), Phe (F) or Nal; X₁₇ is Leu (L), Gly (G) or Nal; X₁₈ isLys (K), Orn, Gln (Q) or Asn (N); X₁₉ is Lys (K), Orn, Gln (Q) or Asn(N); X₂₀ is Lys (K) or Orn; X₂₁ is Leu (L); and X₂₂ is Lys (K) or Orn.27. The ApoA-I agonist compound of claim 26 in which:X₂ is Val (V); X₃is Leu (L); X₅ is Leu (L); X₆ is Phe (F); X₇ is Arg (R) or Lys (K); X₈is Glu (E); X₉ is Leu (L); X₁₀ is Leu (L); X₁₁ is Asn (N) or Glu (Q);X₁₂ is Glu (E); and X₁₅ is Glu (E).
 28. The ApoA-I agonist compound ofclaim 24 in which X₁₃ is Gly (G) and each of X₉, X₁₀, X₁₄, X₁₆ and X₁₇is other than Gly (G).
 29. The ApoA-I agonist compound of claim 24 inwhich:X₁ is Pro (P), Gly (G) or D-Pro (p); X₂ is Val (V); X₃ is Leu (L);X₄ is Asp (D) or Glu (E); X₅ is L (Leu) or Phe (F); X₆ is Phe (F); X₇ isArg (R); X₈ is Glu (E); X₉ is Leu (L); X₁₀ is Leu (L) or Trp (W); X₁₁ isAsn (N); X₁₂ is Glu (E); X₁₃ is Gly (G); X₁₄ is Leu (L); X₁₅ is Glu (E);X₁₆ is Ala (A) or Trp (W); X₁₇ is Leu (L) or Nal; X₁₈ is Lys (K) or Orn;X₁₉ is Gln (Q); X₂₀ is Lys (K) or Orn; X₂₁ is Leu (L); and X₂₂ is Lys(K) or Orn.
 30. The ApoA-I agonist compound of claim 29 which isselected from the group consisting of:

    ______________________________________                                        peptide 3                                                                              PVLDLFRELLNEGLEALKQKLK                                                                          (SEQ ID NO:3);                                     peptide 13        GVLDLFRELLNEGLEALKQKLK                (SEQ ID NO:13);         - peptide 19        pVLDLFRELLNEGLEALKQKLK         (SEQ ID NO:19);            - peptide 137       PVLDLFRELLNEGLEAZKQKLK         (SEQ ID NO:137);           - peptide 138       PVLDLFRELLNEGLEWLKQKLK         (SEQ ID NO:138);           - peptide 139       PVLDLFRELWNEGLEALKQKLK         (SEQ ID NO:139);           - peptide 140       PVLDLFRELLNEGLEALOQOLO         (SEQ ID NO:140);           - peptide 141       PVLDFFRELLNEGLEALKQKLK         (SEQ ID NO:141);           -   and                                                                       - peptide 142       PVLELFRELLNEGLEALKQKLK         (SEQ ID NO:142).         ______________________________________                                    


31. The ApoA-I agonist compound of claim 24 in which X₉ is Gly (G) andeach of X₁₀, X₁₃, X₁₄, X₁₆ and X₁₇ is other than Gly (G).
 32. The ApoA-Iagonist compound of claim 31 which is PVLDLFREGLNELLEALKQKLK (SEQ IDNO:20).
 33. The ApoA-I agonist compound of claim 24 in which X₁₀ is Gly(G) and each of X₉, X₁₃, X₁₄, X₁₆ and X₁₇ is other than Gly (G).
 34. TheApoA-I agonist compound of claim 33 which is PVLDLFRELGNELLEALKQKLK (SEQID NO:9).
 35. The ApoA-I agonist compound of claim 24 in which X₁₄ isGly (G) and each of X₉, X₁₀, X₁₃, X₁₆ and X₁₇ is other than Gly (G). 36.The ApoA-I agonist compound of claim 35 which is PVLDLFRELLNELGEALKQKLK(SEQ ID NO: 126).
 37. The ApoA-I agonist compound of claim 24 in whichX₁₆ is Gly (G) and each of X₉, X₁₀, X₁₃, X₁₄ and X₁₇ is other than Gly(G).
 38. The ApoA-I agonist compound of claim 37 which isPVLDLFRELLNELLEGLKQKLK (SEQ ID NO:22).
 39. The ApoA-I agonist compoundof claim 24 in which X₁₇ is Gly (G) and each of X₉, X₁₀, X₁₃, X₁₄ andX₁₆ is other than Gly (G).
 40. The ApoA-I agonist compound of claim 39which is PVLDLFRELLNELLEAGKQKLK (SEQ ID NO:12).
 41. The pharmaceuticalcomposition of claim 18 in which the ApoA-I agonist compound is apeptide or peptide analogue according to claim
 23. 42. Thepharmaceutical composition of claim 18 in which the ApoA-I agonistcompound is a peptide according to claim
 24. 43. The pharmaceuticalcomposition of claim 18 in which the ApoA-I agonist compound is apeptide according to claim
 25. 44. The pharmaceutical composition ofclaim 18 in which the ApoA-I agonist compound is a peptide according toclaim
 26. 45. The pharmaceutical composition of claim 18 in which theApoA-I agonist compound is a peptide according to claim
 27. 46. Thepharmaceutical composition of claim 18 in which the ApoA-I agonistcompound is a peptide according to claim
 28. 47. The pharmaceuticalcomposition of claim 18 in which the ApoA-I agonist compound is apeptide according to claim
 29. 48. The pharmaceutical composition ofclaim 18 in which the ApoA-I agonist compound is a peptide according toclaim
 30. 49. The pharmaceutical composition of claim 18 in which theApoA-I agonist compound is a peptide according to claim
 31. 50. Thepharmaceutical composition of claim 18 in which the ApoA-I agonistcompound is a peptide according to claim
 32. 51. The pharmaceuticalcomposition of claim 18 in which the ApoA-I agonist compound is apeptide according to claim
 33. 52. The pharmaceutical composition ofclaim 18 in which the ApoA-I agonist compound is a peptide according toclaim
 34. 53. The pharmaceutical composition of claim 18 in which theApoA-I agonist compound is a peptide according to claim
 35. 54. Thepharmaceutical composition of claim 18 in which the ApoA-I agonistcompound is a peptide according to claim
 36. 55. The pharmaceuticalcomposition of claim 18 in which the ApoA-I agonist compound is apeptide according to claim
 37. 56. The pharmaceutical composition ofclaim 18 in which the ApoA-I agonist compound is a peptide according toclaim
 38. 57. The pharmaceutical composition of claim 18 in which theApoA-I agonist compound is a peptide according to claim
 39. 58. Thepharmaceutical composition of claim 18 in which the ApoA-I agonistcompound is a peptide according to claim 40.